Methods of Treating or Preventing Autoimmune Diseases With 2,4-Pyrimidinediamine Compounds

ABSTRACT

The present invention provides methods of treating or preventing autoimmune diseases with 2,4-pyrimidinediamine compounds, as well as methods of treating, preventing or ameliorating symptoms associated with such diseases. Specific examples of autoimmune diseases that can be treated or prevented with the compounds include rheumatoid arthritis and/or its associated symptoms, systemic lups erythematosis and/or its associated symptoms and multiple sclerosis and/or its associated symptoms.

1. CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No. 12/691,657 filed Jan. 21, 2010, which is a continuation of application Ser. No. 11/539,101, filed Oct. 5, 2006, now U.S. Pat. No. 7,825,116 issued Nov. 2, 2010, which is a continuation of application Ser. No. 10/631,029, filed Jul. 29, 2003, now U.S. Pat. No. 7,517,886 issued Apr. 14, 2009, which claims benefit under 35 U.S.C. §119(e) to application Ser. No. 60/399,673 filed Jul. 29, 2002; Ser. No. 60/443,949 filed Jan. 31, 2003 and Ser. No. 60/452,339 filed Mar. 6, 2003, the disclosures of which are incorporated herein by reference in their entirety.

2. FIELD OF THE INVENTION

The present invention relates generally to 2,4-pyrimidinediamine compounds, pharmaceutical compositions comprising the compounds, intermediates and synthetic methods of making the compounds and methods of using the compounds and compositions in a variety of contexts, such as in the treatment or prevention of autoimmune diseases and/or the symptoms associated therewith.

3. BACKGROUND OF THE INVENTION

Crosslinking of Fc receptors, such as the high affinity receptor for IgE (FcεRI) and/or the high affinity receptor for IgG (FcγRI) activates a signaling cascade in mast, basophil and other immune cells that results in the release of chemical mediators responsible for numerous adverse events. For example, such crosslinking leads to the release of preformed mediators of Type I (immediate) anaphylactic hypersensitivity reactions, such as histamine, from storage sites in granules via degranulation. It also leads to the synthesis and release of other mediators, including leukotrienes, prostaglandins and platelet-activating factors (PAFs), that play important roles in inflammatory reactions. Additional mediators that are synthesized and released upon crosslinking Fc receptors include cytokines and nitric oxide.

The signaling cascade(s) activated by crosslinking Fc receptors such as FcεRI and/or FcγRI comprises an array of cellular proteins. Among the most important intracellular signal propagators are the tyrosine kinases. And, an important tyrosine kinase involved in the signal transduction pathways associated with crosslinking the FcεRI and/or FcγRI receptors, as well as other signal transduction cascades, is Syk kinase (see Valent et al., 2002, Intl. J. Hematol. 75(4):257-362 for review).

As the mediators released as a result of FcεRI and FcγRI receptor cross-linking are responsible for, or play important roles in, the manifestation of numerous adverse events, the availability of compounds capable of inhibiting the signaling cascade(s) responsible for their release would be highly desirable. Moreover, owing to the critical role that Syk kinase plays these and other receptor signaling cascade(s), the availability of compounds capable of inhibiting Syk kinase would also be highly desirable.

4. SUMMARY OF THE INVENTION

In one aspect, the present invention provides novel 2,4-pyrimidinediamine compounds that, as will be discussed in more detail below, have myriad biological activities. The compounds generally comprise a 2,4-pyrimidinediamine “core” having the following structure and numbering convention:

The compounds of the invention are substituted at the C2 nitrogen (N2) to form a secondary amine and are optionally further substituted at one or more of the following positions: the C4 nitrogen (N4), the C5 position and/or the C6 position. When substituted at N4, the substituent forms a secondary amine. The substituent at N2, as well as the optional substituents at the other positions, may range broadly in character and physico-chemical properties. For example, the substituent(s) may be a branched, straight-chained or cyclic alkyl, a branched, straight-chained or cyclic heteroalkyl, a mono- or polycyclic aryl a mono- or polycyclic heteroaryl or combinations of these groups. These substituent groups may be further substituted, as will be described in more detail below.

The N2 and/or N4 substituents may be attached directly to their respective nitrogen atoms, or they may be spaced away from their respective nitrogen atoms via linkers, which may be the same or different. The nature of the linkers can vary widely, and can include virtually any combination of atoms or groups useful for spacing one molecular moiety from another. For example, the linker may be an acyclic hydrocarbon bridge (e.g, a saturated or unsaturated alkyleno such as methano, ethano, etheno, propano, prop[1]eno, butano, but[1]eno, but[2]eno, buta[1,3]dieno, and the like), a monocyclic or polycyclic hydrocarbon bridge (e.g., [1,2]benzeno, [2,3]naphthaleno, and the like), a simple acyclic heteroatomic or heteroalkyldiyl bridge (e.g., —O—, —S—, —S—O—, —NH—, —PH—, —C(O)—, —C(O)NH—, —S(O)—, —S(O)₂—, —S(O)NH—, —S(O)₂NH—, —O—CH₂—, —CH₂—O—CH₂—, —O—CH═CH—CH₂—, and the like), a monocyclic or polycyclic heteroaryl bridge (e.g., [3,4]furano, pyridino, thiopheno, piperidino, piperazino, pyrazidino, pyrrolidino, and the like) or combinations of such bridges.

The substituents at the N2, N4, C5 and/or C6 positions, as well as the optional linkers, may be further substituted with one or more of the same or different substituent groups. The nature of these substituent groups may vary broadly. Non-limiting examples of suitable substituent groups include branched, straight-chain or cyclic alkyls, mono- or polycyclic aryls, branched, straight-chain or cyclic heteroalkyls, mono- or polycyclic heteroaryls, halos, branched, straight-chain or cyclic haloalkyls, hydroxyls, oxos, thioxos, branched, straight-chain or cyclic alkoxys, branched, straight-chain or cyclic haloalkoxys, trifluoromethoxys, mono- or polycyclic aryloxys, mono- or polycyclic heteroaryloxys, ethers, alcohols, sulfides, thioethers, sulfanyls (thiols), imines, azos, azides, amines (primary, secondary and tertiary), nitriles (any isomer), cyanates (any isomer), thiocyanates (any isomer), nitrosos, nitros, diazos, sulfoxides, sulfonyls, sulfonic acids, sulfamides, sulfonamides, sulfamic esters, aldehydes, ketones, carboxylic acids, esters, amides, amidines, formadines, amino acids, acetylenes, carbamates, lactones, lactams, glucosides, gluconurides, sulfones, ketals, acetals, thioketals, oximes, oxamic acids, oxamic esters, etc., and combinations of these groups. Substituent groups bearing reactive functionalities may be protected or unprotected, as is well-known in the art.

In one illustrative embodiment, the 2,4-pyrimidinediamine compounds of the invention are compounds according to structural formula (I):

including salts, hydrates, solvates and N-oxides thereof, wherein:

L¹ and L² are each, independently of one another, selected from the group consisting of a direct bond and a linker;

R² is selected from the group consisting of (C1-C6) alkyl optionally substituted with one or more of the same or different R⁸ groups, (C3-C8) cycloalkyl optionally substituted with one or more of the same or different R⁸ groups, cyclohexyl optionally substituted with one or more of the same or different R⁸ groups, 3-8 membered cycloheteroalkyl optionally substituted with one or more of the same or different R⁸ groups, (C5-C15) aryl optionally substituted with one or more of the same or different R⁸ groups, phenyl optionally substituted with one or more of the same or different R⁸ groups and 5-15 membered heteroaryl optionally substituted with one or more of the same or different R⁸ groups;

R⁴ is selected from the group consisting of hydrogen, (C1-C6) alkyl optionally substituted with one or more of the same or different R⁸ groups, (C3-C8) cycloalkyl optionally substituted with one or more of the same or different R⁸ groups, cyclohexyl optionally substituted with one or more of the same or different R⁸ groups, 3-8 membered cycloheteroalkyl optionally substituted with one or more of the same or different R⁸ groups, (C5-C15) aryl optionally substituted with one or more of the same or different R⁸ groups, phenyl optionally substituted with one or more of the same or different R⁸ groups and 5-15 membered heteroaryl optionally substituted with one or more of the same or different R⁸ groups;

R⁵ is selected from the group consisting of R⁶, (C1-C6) alkyl optionally substituted with one or more of the same or different R⁸ groups, (C1-C4) alkanyl optionally substituted with one or more of the same or different R⁸ groups, (C2-C4) alkenyl optionally substituted with one or more of the same or different R⁸ groups and (C2-C4) alkynyl optionally substituted with one or more of the same or different R⁸ groups;

each R⁶ is independently selected from the group consisting of hydrogen, an electronegative group, —OR^(d), —SR^(d), (C1-C3) haloalkyloxy, (C1-C3) perhaloalkyloxy, —NR^(c)R^(c), halogen, (C1-C3) haloalkyl, (C1-C3) perhaloalkyl, —CF₃, —CH₂CF₃, —CF₂CF₃, —CN, —NC, —OCN, —SCN, —NO, —NO₂, —N₃, —S(O)R^(d), —S(O)₂R^(d), —S(O)₂OR^(d), —S(O)NR^(c)R^(c); —S(O)₂NR^(c)R^(c), —OS(O)R^(d), —OS(O)₂R^(d), —OS(O)₂OR^(d), —OS(O)NR^(c)R^(c), —OS(O)₂NR^(c)R^(c), —C(O)R^(d), —C(O)OR^(d), —C(O)NR^(c)R^(c), —C(NH)NR^(c)R^(c), —OC(O)R^(d), —SC(O)R^(d), —OC(O)OR^(d), —SC(O)OR^(d), —OC(O)NR^(c)R^(c), —SC(O)NR^(c)R^(c), —OC(NH)NR^(c)R^(c), —SC(NH)NR^(c)R^(c), —[NHC(O)]_(n)R^(d), —[NHC(O)]_(n)OR^(d), —[NHC(O)]_(n)NR^(c)R^(c) and —[NHC(NH)]_(n)NR^(c)R^(c), (C5-C10) aryl optionally substituted with one or more of the same or different R⁸ groups, phenyl optionally substituted with one or more of the same or different R⁸ groups, (C6-C16) arylalkyl optionally substituted with one or more of the same or different R⁸ groups, 5-10 membered heteroaryl optionally substituted with one or more of the same or different R⁸ groups and 6-16 membered heteroarylalkyl optionally substituted with one or more of the same or different R⁸ groups;

R⁸ is selected from the group consisting of R^(a), R^(b), R^(a) substituted with one or more of the same or different R^(a) or R^(b), —OR^(a) substituted with one or more of the same or different R^(a) or R^(b), —B(OR^(a))₂, —B(NR^(c)R^(c))₂, —(CH₂)_(m)—R^(b), —(CHR^(a))_(m)—R^(b), —O—(CH₂)_(m)—R^(b), —S—(CH₂)_(m)—R^(b), —O—CHR^(a)R^(b), —O—CR^(a)(R^(b))₂, —O—(CHR^(a))_(m)—R^(b), —O— (CH₂)_(m)—CH[(CH₂)_(m)R^(b)]R^(b), —S—(CHR^(a))_(m)—R^(b), —C(O)NH—(CH₂)_(m)—R^(b), —C(O)NH—(CHR^(a))_(m)—R^(b), —O—(CH₂)_(m)—C(O)NH—(CH₂)_(m)—R^(b), —S—(CH₂)_(m)—C(O)NH—(CH₂)_(m)—R^(b), —O—(CHR^(a))_(m)—C(O)NH—(CHR^(a))_(m)—R^(b), —S—(CHR^(a))_(m)—C(O)NH—(CHR^(a))_(m)—R^(b), —NH—(CH₂)_(m)—R^(b), —NH—(CHR^(a))_(m)—R^(b), —NH[(CH₂)_(m)R^(b)], —N[(CH₂)_(m)R^(b)]₂, —NH—C(O)—NH—(CH₂)_(m)—R^(b), —NH—C(O)—(CH₂)_(m)—CHR^(b)R^(b) and —NH—(CH₂)_(m)—C(O)—NH—(CH₂)_(m)—R^(b);

each R^(a) is independently selected from the group consisting of hydrogen, (C1-C6) alkyl, (C3-C8) cycloalkyl, cyclohexyl, (C4-C11) cycloalkylalkyl, (C5-C10) aryl, phenyl, (C6-C16) arylalkyl, benzyl, 2-6 membered heteroalkyl, 3-8 membered cycloheteroalkyl, morpholinyl, piperazinyl, homopiperazinyl, piperidinyl, 4-11 membered cycloheteroalkylalkyl, 5-10 membered heteroaryl and 6-16 membered heteroarylalkyl;

each R^(b) is a suitable group independently selected from the group consisting of ═O, —OR^(d), (C1-C3) haloalkyloxy, —OCF₃, ═S, —SR^(d), ═NR^(d), ═NOR^(d), —NR^(c)R^(c), halogen, —CF₃, —CN, —NC, —OCN, —SCN, —NO, —NO₂, ═N₂, —N₃, —S(O)R^(d), —S(O)₂R^(d), —S(O)₂OR^(d), —S(O)NR^(c)R^(c), —S(O)₂NR^(c)R^(c), —OS(O)R^(d), —OS(O)₂R^(d), —OS(O)₂OR^(d), —OS(O)₂NR^(c)R^(c), —C(O)R^(d), —C(O)OR^(d), —C(O)NR^(c)R^(c), —C(NH)NR^(c)R^(c), —C(NR^(a))NR^(c)R^(c), —C(NOH)R^(a), —C(NOH)NR^(c)R^(c), —OC(O)R^(d), —OC(O)OR^(d), —OC(O)NR^(c)R^(c), —OC(NH)NR^(c)R^(c), —OC(NR^(a))NR^(c)R^(c), —[NHC(O)]_(n)R^(d), —[NR^(a)C(O)]_(n)R^(d), —[NHC(O)]_(n)OR^(d), —[NR^(a)C(O)]_(n)OR^(d), —[NHC(O)]₂NR^(c)R^(c), —[NR^(a)C(O)]₂NR^(c)R^(c), —[NHC(NH)]_(n)NR^(c)R^(c) and —[NR^(a)C(NR^(a))]_(n)NR^(c)R^(c);

each R^(c) is independently a protecting group or R^(a), or, alternatively, each R^(c) is taken together with the nitrogen atom to which it is bonded to form a 5 to 8-membered cycloheteroalkyl or heteroaryl which may optionally include one or more of the same or different additional heteroatoms and which may optionally be substituted with one or more of the same or different R^(a) or suitable R^(b) groups;

each R^(d) is independently a protecting group or R^(a);

each m is independently an integer from 1 to 3; and

each n is independently an integer from 0 to 3.

In another aspect, the present invention provides prodrugs of the 2,4-pyrimidinediamine compounds. Such prodrugs may be active in their prodrug form, or may be inactive until converted under physiological or other conditions of use to an active drug form. In the prodrugs of the invention, one or more functional groups of the 2,4-pyrimidinediamine compounds are included in promoieties that cleave from the molecule under the conditions of use, typically by way of hydrolysis, enzymatic cleavage or some other cleavage mechanism, to yield the functional groups. For example, primary or secondary amino groups may be included in an amide promoiety that cleaves under conditions of use to generate the primary or secondary amino group. Thus, the prodrugs of the invention include special types of protecting groups, termed “progroups,” masking one or more functional groups of the 2,4-pyrimidinediamine compounds that cleave under the conditions of use to yield an active 2,4-pyrimidinediamine drug compound. Functional groups within the 2,4-pyrimidinediamine compounds that may be masked with progroups for inclusion in a promoiety include, but are not limited to, amines (primary and secondary), hydroxyls, sulfanyls (thiols), carboxyls, carbonyls, phenols, catechols, diols, alkynes, phosphates, etc. Myriad progroups suitable for masking such functional groups to yield promoieties that are cleavable under the desired conditions of use are known in the art. All of these progroups, alone or in combinations, may be included in the prodrugs of the invention. Specific examples of promoieties that yield primary or secondary amine groups that can be included in the prodrugs of the invention include, but are not limited to amides, carbamates, imines, ureas, phosphenyls, phosphoryls and sulfenyls. Specific examples of promoieties that yield sulfanyl groups that can be included in the prodrugs of the invention include, but are not limited to, thioethers, for example S-methyl derivatives (monothio, dithio, oxythio, aminothio acetals), silyl thioethers, thioesters, thiocarbonates, thiocarbamates, asymmetrical disulfides, etc. Specific examples of promoieties that cleave to yield hydroxyl groups that can be included in the prodrugs of the invention include, but are not limited to, sulfonates, esters and carbonates. Specific examples of promoieties that yield carboxyl groups that can be included in the prodrugs of the invention included, but are not limited to, esters (including silyl esters, oxamic acid esters and thioesters), amides and hydrazides.

In one illustrative embodiment, the prodrugs of the invention are compounds according to structural formula (I) in which the protecting group of R^(c) and R^(d) is a progroup.

Replacing the hydrogens attached to N2 and N4 in the 2,4-pyrimidinediamines of structural formula (I) with substituents adversely affects the activity of the compounds. However, as will be appreciated by skilled artisans, these nitrogens may be included in promoieties that, under conditions of use, cleave to yield 2,4-pyrimidinediamines according to structural formula (I). Thus, in another illustrative embodiment, the prodrugs of the invention are compounds according to structural formula (II):

including salts, hydrates, solvates and N-oxides thereof, wherein:

R², R⁴, R⁵, R⁶, L¹ and L² are as previously defined for structural formula (I); and

R^(2b) and R^(4b) are each, independently of one another, a progroup.

In another aspect, the present invention provides compositions comprising one or more compounds and/or prodrugs of the invention and an appropriate carrier, excipient or diluent. The exact nature of the carrier, excipient or diluent will depend upon the desired use for the composition, and may range from being suitable or acceptable for veterinary uses to being suitable or acceptable for human use.

In still another aspect, the present invention provides intermediates useful for synthesizing the 2,4-pyrimidinediamine compounds and prodrugs of the invention. In one embodiment, the intermediates are 4-pyrimidineamines according to structural formula (III):

including salts, hydrates, solvates and N-oxides thereof, wherein R⁴, R⁵, R⁶ and L² are as previously defined for structural formula (I); LG is a leaving group such as, for example, —S(O)₂Me, —SMe or halo (e.g., F, Cl, Br, I); and R^(4c) is hydrogen or a progroup.

In another embodiment, the intermediates are 2-pyrimidineamines according to structural formula (IV):

including salts, hydrates, solvates and N-oxides thereof, wherein R², R⁵, R⁶ and L¹ are as previously defined for structural formula (I); LG is a leaving group, such as, for example, —S(O)₂Me, —SMe or halo (e.g., F, Cl, Br, I) and R^(2c) is hydrogen or a progroup.

In yet another embodiment, the intermediates are 4-amino- or 4-hydroxy-2-pyrimidineamines according to structural formula (V):

including salts, hydrates, solvates and N-oxides thereof, wherein R², R⁵, R⁶ and L¹ are as previously defined for structural formula (I), R⁷ is an amino or hydroxyl group and R^(2c) is hydrogen or a progroup.

In another embodiment, the intermediates are N4-substituted cytosines according to structural formula (VI):

including salts, hydrates, solvates and N-oxides thereof, wherein R⁴, R⁵, R⁶ and L² are as previously defined for structural formula (I) and R^(4c) is hydrogen or a progroup.

In yet another aspect, the present invention provides methods of synthesizing the 2,4-pyrimidinediamine compounds and prodrugs of the invention. In one embodiment, the method involves reacting a 4-pyrimidineamine according to structural formula (III) with an amine of the formula HR^(2c)N-L¹-R², where L¹, R² and R^(2c) are as previously defined for structural formula (IV) to yield a 2,4-pyrimidinediamine according to structural formula (I) or a prodrug according to structural formula (II).

In another embodiment, the method involves reacting a 2-pyrimidineamine according to structural formula (IV) with an amine of the formula R⁴-L²-NHR^(4c) where L⁴, R⁴ and R^(4c) are as previously defined for structural formula (III) to yield a 2,4-pyrimidinediamine according to structural formula (I) or a prodrug according to structural formula (II).

In yet another embodiment, the method involves reacting a 4-amino-2-pyrimidineamine according to structural formula (V) (in which R⁷ is an amino group) with an amine of the formula R⁴-L²-NHR^(4c), where L², R⁴ and R^(4c) are as defined for structural formula (III), to yield a 2,4-pyrimidinediamine according to structural formula (I) or a prodrug according to structural formula (II). Alternatively, the 4-amino-2-pyrimidineamine may be reacted with a compound of the formula R⁴-L²-LG, where R⁴ and L² are as previously defined for structural formula (I) and LG is a leaving group.

In still another embodiment, the method involves halogenating a 4-hydroxy-2-pyrimidineamine according to structural formula (V) (R⁷ is a hydroxyl group) to yield a 2-pyrimidineamine according to structural formula (IV) and reacting this pyrimidineamine with an appropriate amine, as described above.

In yet another embodiment, the method involves halogenating an N4-substituted cytosine according to structural formula (VI) to yield a 4-pyrimidineamine according to structural formula (III) and reacting this pyrimidineamine with an appropriate amine, as described above.

The 2,4-pyrimidinediamine compounds of the invention are potent inhibitors of degranulation of immune cells, such as mast, basophil, neutrophil and/or eosinophil cells. Thus, in still another aspect, the present invention provides methods of regulating, and in particular inhibiting, degranulation of such cells. The method generally involves contacting a cell that degranulates with an amount of a 2,4-pyrimidinediamine compound or prodrug of the invention, or an acceptable salt, hydrate, solvate, N-oxide and/or composition thereof, effective to regulate or inhibit degranulation of the cell. The method may be practiced in in vitro contexts or in in vivo contexts as a therapeutic approach towards the treatment or prevention of diseases characterized by, caused by or associated with cellular degranulation.

While not intending to be bound by any theory of operation, biochemical data confirm that the 2,4-pyrimidinediamine compounds exert their degranulation inhibitory effect, at least in part, by blocking or inhibiting the signal transduction cascade(s) initiated by crosslinking of the high affinity Fc receptors for IgE (“FcεRI”) and/or IgG (“FcγRI”). Indeed, the 2,4-pyrimidinediamine compounds are potent inhibitors of both FcεRI-mediated and FcγRI-mediated degranulation. As a consequence, the 2,4-pyrimidine compounds may be used to inhibit these Fc receptor signalling cascades in any cell type expressing such FcεRI and/or FcγRI receptors including but not limited to macrophages, mast, basophil, neutrophil and/or eosinophil cells.

The methods also permit the regulation of, and in particular the inhibition of, downstream processes that result as a consequence of activating such Fc receptor signaling cascade(s). Such downstream processes include, but are not limited to, FcεRI-mediated and/or FcγRI-mediated degranulation, cytokine production and/or the production and/or release of lipid mediators such as leukotrienes and prostaglandins. The method generally involves contacting a cell expressing an Fc receptor, such as one of the cell types discussed above, with an amount of a 2,4-pyrimidinediamine compound or prodrug of the invention, or an acceptable salt, hydrate, solvent, N-oxide and/or composition thereof, effective to regulate or inhibit the Fc receptor signaling cascade and/or a downstream process effected by the activation of this signaling cascade. The method may be practiced in in vitro contexts or in in vivo contexts as a therapeutic approach towards the treatment or prevention of diseases characterized by, caused by or associated with the Fc receptor signaling cascade, such as diseases effected by the release of granule specific chemical mediators upon degranulation, the release and/or synthesis of cytokines and/or the release and/or synthesis of lipid mediators such as leukotrienes and prostaglandins.

In yet another aspect, the present invention provides methods of treating and/or preventing diseases characterized by, caused by or associated with the release of chemical mediators as a consequence of activating Fc receptor signaling cascades, such as FcεRI and/or FcγRI-signaling cascades. The methods may be practiced in animals in veterinary contexts or in humans. The methods generally involve administering to an animal subject or human an amount of a 2,4-pyrimidinediamine compound or prodrug of the invention, or an acceptable salt, hydrate, solvate, N-oxide and/or composition thereof, effective to treat or prevent the disease. As discussed previously, activation of the FcεRI or FcγRI receptor signaling cascade in certain immune cells leads to the release and/or synthesis of a variety of chemical substances that are pharmacological mediators of a wide variety of diseases. Any of these diseases may be treated or prevented according to the methods of the invention.

For example, in mast cells and basophil cells, activation of the FcεRI or FcγRI signaling cascade leads to the immediate (i.e., within 1-3 min. of receptor activation) release of preformed mediators of atopic and/or Type I hypersensitivity reactions (e.g., histamine, proteases such as tryptase, etc.) via the degranulation process. Such atopic or Type I hypersensitivity reactions include, but are not limited to, anaphylactic reactions to environmental and other allergens (e.g., pollens, insect and/or animal venoms, foods, drugs, contrast dyes, etc.), anaphylactoid reactions, hay fever, allergic conjunctivitis, allergic rhinitis, allergic asthma, atopic dermatitis, eczema, urticaria, mucosal disorders, tissue disorders and certain gastrointestinal disorders.

The immediate release of the preformed mediators via degranulation is followed by the release and/or synthesis of a variety of other chemical mediators, including, among other things, platelet activating factor (PAF), prostaglandins and leukotrienes (e.g., LTC4) and the de novo synthesis and release of cytokines such as TNFα, IL-4, IL-5, IL-6, IL-13, etc. The first of these two processes occurs approximately 3-30 min. following receptor activation; the latter approximately 30 min.-7 hrs. following receptor activation. These “late stage” mediators are thought to be in part responsible for the chronic symptoms of the above-listed atopic and Type I hypersensitivity reactions, and in addition are chemical mediators of inflammation and inflammatory diseases (e.g., osteoarthritis, inflammatory bowel disease, ulcerative colitis, Crohn's disease, idiopathic inflammatory bowel disease, irritable bowel syndrome, spastic colon, etc.), low grade scarring (e.g., scleroderma, increased fibrosis, keloids, post-surgical scars, pulmonary fibrosis, vascular spasms, migraine, reperfusion injury and post myocardial infarction), and sicca complex or syndrome. All of these diseases may be treated or prevented according to the methods of the invention.

Additional diseases which can be treated or prevented according to the methods of the invention include diseases associated with basophil cell and/or mast cell pathology. Examples of such diseases include, but are not limited to, diseases of the skin such as scleroderma, cardiac diseases such as post myocardial infarction, pulmonary diseases such as pulmonary muscle changes or remodeling and chronic obstructive pulmonary disease (COPD) and diseases of the gut such as inflammatory bowel syndrome (spastic colon).

The 2,4-pyrimidinediamine compounds of the invention are also potent inhibitors of the tyrosine kinase Syk kinase. Thus, in still another aspect, the present invention provides methods of regulating, and in particular inhibiting, Syk kinase activity. The method generally involves contacting a Syk kinase or a cell comprising a Syk kinase with an amount of a 2,4-pyrimidinediamine compound or prodrug of the invention, or an acceptable salt, hydrate, solvate, N-oxide and/or composition thereof, effective to regulate or inhibit Syk kinase activity. In one embodiment, the Syk kinase is an isolated or recombinant Syk kinase. In another embodiment, the Syk kinase is an endogenous or recombinant Syk kinase expressed by a cell, for example a mast cell or a basophil cell. The method may be practiced in in vitro contexts or in in vivo contexts as a therapeutic approach towards the treatment or prevention of diseases characterized by, caused by or associated with Syk kinase activity.

While not intending to be bound by any particular theory of operation, it is believed that the 2,4-pyrimdinediamine compounds of the invention inhibit cellular degranulation and/or the release of other chemical mediators primarily by inhibiting Syk kinase that gets activated through the gamma chain homodimer of FcεRI (see, e.g., FIG. 2). This gamma chain homodimer is shared by other Fc receptors, including FcγRI, FcγRIII and FcαRI. For all of these receptors, intracellular signal transduction is mediated by the common gamma chain homodimer. Binding and aggregation of those receptors results in the recruitment and activation of tyrosine kinases such as Syk kinase. As a consequence of these common signaling activities, the 2,4-pyrimidinediamine compounds described herein may be used to regulate, and in particular inhibit, the signaling cascades of Fc receptors having this gamma chain homodimer, such as FcεRI, FcγRI, FcγRIII and FcαRI, as well as the cellular responses elicited through these receptors.

Syk kinase is known to play a critical role in other signaling cascades. For example, Syk kinase is an effector of B-cell receptor (BCR) signaling (Turner et al., 2000, Immunology Today 21:148-154) and is an essential component of integrin beta(1), beta(2) and beta(3) signaling in neutrophils (Mocsai et al., 2002, Immunity 16:547-558). As the 2,4-pyrimidinediamine compounds described herein are potent inhibitors of Syk kinase, they can be used to regulate, and in particular inhibit, any signaling cascade where Syk plays a role, such as, fore example, the Fc receptor, BCR and integrin signaling cascades, as well as the cellular responses elicited through these signaling cascades. The particular cellular response regulated or inhibited will depend, in part, on the specific cell type and receptor signaling cascade, as is well known in the art. Non-limiting examples of cellular responses that may be regulated or inhibited with the 2,4-pyrimidinediamine compounds include a respiratory burst, cellular adhesion, cellular degranulation, cell spreading, cell migration, phagocytosis (e.g., in macrophages), calcium ion flux (e.g., in mast, basophil, neutrophil, eosinophil and B-cells), platelet aggregation, and cell maturation (e.g., in B-cells).

Thus, in another aspect, the present invention provides methods of regulating, and in particular inhibiting, signal transduction cascades in which Syk plays a role. The method generally involves contacting a Syk-dependent receptor or a cell expressing a Syk-dependent receptor with an amount of a 2,4-pyrimidinediamine compound or prodrug of the invention, or an acceptable salt, hydrate, solvate, N-oxide and/or composition thereof, effective to regulate or inhibit the signal transduction cascade. The methods may also be used to regulate, and in particular inhibit, downstream processes or cellular responses elicited by activation of the particular Syk-dependent signal transduction cascade. The methods may be practiced to regulate any signal transduction cascade where Syk is not known or later discovered to play a role. The methods may be practiced in in vitro contexts or in in vivo contexts as a therapeutic approach towards the treatment or prevention of diseases characterized by, caused by or associated with activation of the Syk-dependent signal transduction cascade. Non-limited examples of such diseases include those previously discussed.

Cellular and animal data also confirm that the 2,4-pyrimidinediamine compounds of the invention may also be used to treat or prevent autoimmune diseases and/or symptoms of such diseases. The methods generally involve administering to a subject suffering from an autoimmune disease or at risk of developing an autoimmune disease an amount of a 2,4-pyrimidinediamine method or prodrug of the invention, or an acceptable salt, N-oxide, hydrate, solvate or composition thereof, effective to treat or prevent the autoimmune disease and/or its associated symptoms. Autoimmune diseases that can be treated or prevented with the 2,4-pyrimidinediamine compounds include those diseases that are commonly associated with nonanaphylactic hypersensitivity reactions (Type II, Type III and/or Type IV hypersensitivity reactions) and/or those diseases that are mediated, at least in part, by activation of the FcγR signaling cascade in monocyte cells. Such autoimmune disease include, but are not limited to, those autoimmune diseases that are frequently designated as single organ or single cell-type autoimmune disorders and those autoimmune disease that are frequently designated as involving systemic autoimmune disorder. Non-limiting examples of diseases frequently designated as single organ or single cell-type autoimmune disorders include: Hashimoto's thyroiditis, autoimmune hemolytic anemia, autoimmune atrophic gastritis of pernicious anemia, autoimmune encephalomyelitis, autoimmune orchitis, Goodpasture's disease, autoimmune thrombocytopenia, sympathetic ophthalmia, myasthenia gravis, Graves' disease, primary biliary cirrhosis, chronic aggressive hepatitis, ulcerative colitis and membranous glomerulopathy. Non-limiting examples of diseases often designated as involving systemic autoimmune disorder include: systemic lupus erythematosis, rheumatoid arthritis, Sjogren's syndrome, Reiter's syndrome, polymyositis-dermatomyositis, systemic sclerosis, polyarteritis nodosa, multiple sclerosis and bullous pemphigoid.

5. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 provides a cartoon illustrating allergen-induced production of IgE and consequent release of preformed and other chemical mediators from mast cells;

FIG. 2 provides a cartoon illustrating the FcεR1 signal transduction cascade leading to degranulation of mast and/or basophil cells;

FIG. 3 provides a cartoon illustrating the putative points of action of compounds that selectively inhibit upstream FcεRI-mediated degranulation and compounds that inhibit both FcεRI-mediated and ionomycin-induced degranulation;

FIG. 4 provides graphs illustrating the effects of certain 2,4-pyrimidinediamine compounds, DMSO (control) and ionomycin on Ca²⁺ flux in CHMC cells;

FIG. 5 provides graphs illustrating the immediacy of the inhibitory activity of compounds R921218 and R926495;

FIG. 6 provides a graph illustrating the effect of washout on the inhibitory activity of compounds R921218 and R921302;

FIG. 7 provides data showing that varying concentrations of compounds R921218 (A) and R921219 (B) inhibit phosphorylation of various proteins downstream of Syk kinase in the IgE receptor signal transduction cascade in activated BMMC cells;

FIG. 8 provides data showing dose responsive inhibition of Syk kinase phosphorylation of an endogenous substrate (LAT) and a peptide substrate in the presence of increasing concentrations of compounds R921218 (X), R921219 (Y) and R921304 (Z);

FIG. 9 provides data showing that the inhibition of Syk kinase by compound R921219 is ATP competitive;

FIG. 10 provides data showing that varying concentrations of compounds R921219 (A) and R218218 (B) inhibit phosphorylation of proteins downstream of Syk kinase, but not LYN kinase, in the FcεRI signal transduction cascade in activated CHMC cells; also shown is inhibition of phosphorylation of proteins downstream of LYN kinase but not Syk kinase, in the presence of a known LYN kinase inhibitor (PP2);

FIGS. 11A-D provide data showing inhibition of phosphorylation of proteins downstream of Syk kinase in the FcεRI signal transduction cascade in BMMC cells;

FIG. 12 is a graph illustrating the efficacy of compound R921302 in a mouse model of collagen antibody-induced arthritis (“CAIA”);

FIG. 13 is a graph illustrating the efficacy of compound R921302 in the CAIA model as compared to other agents and control agents;

FIG. 14 is a graph illustrating the efficacy of compound R921302 in a rat model of collagen-induced arthritis (“CIA”);

FIG. 15 is a graph illustrating the efficacy of compound R921302 in inhibiting experimental autoimmune encephalomyelitis (“EAE”) in mice, a clinical model for multiple sclerosis; and

FIG. 16 is a graph illustrating the efficacy compound R921302 on SJL mice treated on the starting day of immunization with 150 μg PLP 139-151/200 μg MTB (CFA).

6. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 6.1 Definitions

As used herein, the following terms are intended to have the following meanings:

“Alkyl” by itself or as part of another substituent refers to a saturated or unsaturated branched, straight-chain or cyclic monovalent hydrocarbon radical having the stated number of carbon atoms (i.e., C1-C6 means one to six carbon atoms) that is derived by the removal of one hydrogen atom from a single carbon atom of a parent alkane, alkene or alkyne. Typical alkyl groups include, but are not limited to, methyl; ethyls such as ethanyl, ethenyl, ethynyl; propyls such as propan-1-yl, propan-2-yl, cyclopropan-1-yl, prop-1-en-1-yl, prop-1-en-2-yl, prop-2-en-1-yl, cycloprop-1-en-1-yl; cycloprop-2-en-1-yl, prop-1-yn-1-yl, prop-2-yn-1-yl, etc.; butyls such as butan-1-yl, butan-2-yl, 2-methyl-propan-1-yl, 2-methyl-propan-2-yl, cyclobutan-1-yl, but-1-en-1-yl, but-1-en-2-yl, 2-methyl-prop-1-en-1-yl, but-2-en-1-yl, but-2-en-2-yl, buta-1,3-dien-1-yl, buta-1,3-dien-2-yl, cyclobut-1-en-1-yl, cyclobut-1-en-3-yl, cyclobuta-1,3-dien-1-yl, but-1-yn-1-yl, but-1-yn-3-yl, but-3-yn-1-yl, etc.; and the like. Where specific levels of saturation are intended, the nomenclature “alkanyl,” “alkenyl” and/or “alkynyl” is used, as defined below. In preferred embodiments, the alkyl groups are (C1-C6) alkyl.

“Alkanyl” by itself or as part of another substituent refers to a saturated branched, straight-chain or cyclic alkyl derived by the removal of one hydrogen atom from a single carbon atom of a parent alkane. Typical alkanyl groups include, but are not limited to, methanyl; ethanyl; propanyls such as propan-1-yl, propan-2-yl (isopropyl), cyclopropan-1-yl, etc.; butanyls such as butan-1-yl, butan-2-yl (sec-butyl), 2-methyl-propan-1-yl (isobutyl), 2-methyl-propan-2-yl (t-butyl), cyclobutan-1-yl, etc.; and the like. In preferred embodiments, the alkanyl groups are (C1-C6) alkanyl.

“Alkenyl” by itself or as part of another substituent refers to an unsaturated branched, straight-chain or cyclic alkyl having at least one carbon-carbon double bond derived by the removal of one hydrogen atom from a single carbon atom of a parent alkene. The group may be in either the cis or trans conformation about the double bond(s). Typical alkenyl groups include, but are not limited to, ethenyl; propenyls such as prop-1-en-1-yl, prop-1-en-2-yl, prop-2-en-1-yl, prop-2-en-2-yl, cycloprop-1-en-1-yl; cycloprop-2-en-1-yl; butenyls such as but-1-en-1-yl, but-1-en-2-yl, 2-methyl-prop-1-en-1-yl, but-2-en-1-yl, but-2-en-2-yl, buta-1,3-dien-1-yl, buta-1,3-dien-2-yl, cyclobut-1-en-1-yl, cyclobut-1-en-3-yl, cyclobuta-1,3-dien-1-yl, etc.; and the like. In preferred embodiments, the alkenyl group is (C2-C6) alkenyl.

“Alkynyl” by itself or as part of another substituent refers to an unsaturated branched, straight-chain or cyclic alkyl having at least one carbon-carbon triple bond derived by the removal of one hydrogen atom from a single carbon atom of a parent alkyne. Typical alkynyl groups include, but are not limited to, ethynyl; propynyls such as prop-1-yn-1-yl , prop-2-yn-1-yl, etc.; butynyls such as but-1-yn-1-yl, but-1-yn-3-yl, but-3-yn-1-yl, etc.; and the like. In preferred embodiments, the alkynyl group is (C2-C6) alkynyl.

“Alkyldiyl” by itself or as part of another substituent refers to a saturated or unsaturated, branched, straight-chain or cyclic divalent hydrocarbon group having the stated number of carbon atoms (i.e., C1-C6 means from one to six carbon atoms) derived by the removal of one hydrogen atom from each of two different carbon atoms of a parent alkane, alkene or alkyne, or by the removal of two hydrogen atoms from a single carbon atom of a parent alkane, alkene or alkyne. The two monovalent radical centers or each valency of the divalent radical center can form bonds with the same or different atoms. Typical alkyldiyl groups include, but are not limited to, methandiyl; ethyldiyls such as ethan-1,1-diyl, ethan-1,2-diyl, ethen-1,1-diyl, ethen-1,2-diyl; propyldiyls such as propan-1,1-diyl, propan-1,2-diyl, propan-2,2-diyl, propan-1,3-diyl, cyclopropan-1,1-diyl, cyclopropan-1,2-diyl, prop-1-en-1,1-diyl, prop-1-en-1,2-diyl, prop-2-en-1,2-diyl, prop-1-en-1,3-diyl, cycloprop-1-en-1,2-diyl, cycloprop-2-en-1,2-diyl, cycloprop-2-en-1,1-diyl, prop-1-yn-1,3-diyl, etc.; butyldiyls such as, butan-1,1-diyl, butan-1,2-diyl, butan-1,3-diyl, butan-1,4-diyl, butan-2,2-diyl, 2-methyl-propan-1,1-diyl, 2-methyl-propan-1,2-diyl, cyclobutan-1,1-diyl; cyclobutan-1,2-diyl, cyclobutan-1,3-diyl, but-1-en-1,1-diyl, but-1-en-1,2-diyl, but-1-en-1,3-diyl, but-1-en-1,4-diyl, 2-methyl-prop-1-en-1,1-diyl, 2-methanylidene-propan-1,1-diyl, buta-1,3-dien-1,1-diyl, buta-1,3-dien-1,2-diyl, buta-1,3-dien-1,3-diyl, buta-1,3-dien-1,4-diyl, cyclobut-1-en-1,2-diyl, cyclobut-1-en-1,3-diyl, cyclobut-2-en-1,2-diyl, cyclobuta-1,3-dien-1,2-diyl, cyclobuta-1,3-dien-1,3-diyl, but-1-yn-1,3-diyl, but-1-yn-1,4-diyl, buta-1,3-diyn-1,4-diyl, etc.; and the like. Where specific levels of saturation are intended, the nomenclature alkanyldiyl, alkenyldiyl and/or alkynyldiyl is used. Where it is specifically intended that the two valencies are on the same carbon atom, the nomenclature “alkylidene” is used. In preferred embodiments, the alkyldiyl group is (C1-C6) alkyldiyl. Also preferred are saturated acyclic alkanyldiyl groups in which the radical centers are at the terminal carbons, e.g., methandiyl (methano); ethan-1,2-diyl (ethano); propan-1,3-diyl (propano); butan-1,4-diyl (butano); and the like (also referred to as alkylenos, defined infra).

“Alkyleno” by itself or as part of another substituent refers to a straight-chain saturated or unsaturated alkyldiyl group having two terminal monovalent radical centers derived by the removal of one hydrogen atom from each of the two terminal carbon atoms of straight-chain parent alkane, alkene or alkyne. The locant of a double bond or triple bond, if present, in a particular alkyleno is indicated in square brackets. Typical alkyleno groups include, but are not limited to, methano; ethylenos such as ethano, etheno, ethyno; propylenos such as propano, prop[1]eno, propa[1,2]dieno, prop[1]yno, etc.; butylenos such as butano, but[1]eno, but[2]eno, buta[1,3]dieno, but[1]yno, but[2]yno, buta[1,3]diyno, etc.; and the like. Where specific levels of saturation are intended, the nomenclature alkano, alkeno and/or alkyno is used. In preferred embodiments, the alkyleno group is (C1-C6) or (C1-C3) alkyleno. Also preferred are straight-chain saturated alkano groups, e.g., methano, ethano, propano, butano, and the like.

“Heteroalkyl,” Heteroalkanyl,” Heteroalkenyl,” Heteroalkynyl,” Heteroalkyldiyl” and “Heteroalkyleno” by themselves or as part of another substituent refer to alkyl, alkanyl, alkenyl, alkynyl, alkyldiyl and alkyleno groups, respectively, in which one or more of the carbon atoms are each independently replaced with the same or different heteroatoms or heteroatomic groups. Typical heteroatoms and/or heteroatomic groups which can replace the carbon atoms include, but are not limited to, —O—, —S—, —S—O—, —NR′—, —PH—, —S(O)—, —S(O)₂—, —S(O)NR′—, —S(O)₂NR′—, and the like, including combinations thereof, where each R′ is independently hydrogen or (C1-C6) alkyl.

“Cycloalkyl” and “Heterocycloalkyl” by themselves or as part of another substituent refer to cyclic versions of “alkyl” and “heteroalkyl” groups, respectively. For heteroalkyl groups, a heteroatom can occupy the position that is attached to the remainder of the molecule. Typical cycloalkyl groups include, but are not limited to, cyclopropyl; cyclobutyls such as cyclobutanyl and cyclobutenyl; cyclopentyls such as cyclopentanyl and cyclopentenyl; cyclohexyls such as cyclohexanyl and cyclohexenyl; and the like. Typical heterocycloalkyl groups include, but are not limited to, tetrahydrofuranyl (e.g., tetrahydrofuran-2-yl, tetrahydrofuran-3-yl, etc.), piperidinyl (e.g., piperidin-1-yl, piperidin-2-yl, etc.), morpholinyl (e.g., morpholin-3-yl, morpholin-4-yl, etc.), piperazinyl (e.g., piperazin-1-yl, piperazin-2-yl, etc.), and the like.

“Acyclic Heteroatomic Bridge” refers to a divalent bridge in which the backbone atoms are exclusively heteroatoms and/or heteroatomic groups. Typical acyclic heteroatomic bridges include, but are not limited to, —O—, —S—, —S—O—, —NR′—, —PH—, —S(O)—, —S(O)₂—, —S(O)NR′—, —S(O)₂NR′—, and the like, including combinations thereof, where each R′ is independently hydrogen or (C1-C6) alkyl.

“Parent Aromatic Ring System” refers to an unsaturated cyclic or polycyclic ring system having a conjugated π electron system. Specifically included within the definition of “parent aromatic ring system” are fused ring systems in which one or more of the rings are aromatic and one or more of the rings are saturated or unsaturated, such as, for example, fluorene, indane, indene, phenalene, tetrahydronaphthalene, etc. Typical parent aromatic ring systems include, but are not limited to, aceanthrylene, acenaphthylene, acephenanthrylene, anthracene, azulene, benzene, chrysene, coronene, fluoranthene, fluorene, hexacene, hexaphene, hexylene, indacene, s-indacene, indane, indene, naphthalene, octacene, octaphene, octalene, ovalene, penta-2,4-diene, pentacene, pentalene, pentaphene, perylene, phenalene, phenanthrene, picene, pleiadene, pyrene, pyranthrene, rubicene, tetrahydronaphthalene, triphenylene, trinaphthalene, and the like, as well as the various hydro isomers thereof

“Aryl” by itself or as part of another substituent refers to a monovalent aromatic hydrocarbon group having the stated number of carbon atoms (i.e., C5-C15 means from 5 to 15 carbon atoms) derived by the removal of one hydrogen atom from a single carbon atom of a parent aromatic ring system. Typical aryl groups include, but are not limited to, groups derived from aceanthrylene, acenaphthylene, acephenanthrylene, anthracene, azulene, benzene, chrysene, coronene, fluoranthene, fluorene, hexacene, hexaphene, hexylene, as-indacene, s-indacene, indane, indene, naphthalene, octacene, octaphene, octalene, ovalene, penta-2,4-diene, pentacene, pentalene, pentaphene, perylene, phenalene, phenanthrene, picene, pleiadene, pyrene, pyranthrene, rubicene, triphenylene, trinaphthalene, and the like, as well as the various hydro isomers thereof. In preferred embodiments, the aryl group is (C5-C15) aryl, with (C5-C10) being even more preferred. Particularly preferred aryls are cyclopentadienyl, phenyl and naphthyl.

“Arylaryl” by itself or as part of another substituent refers to a monovalent hydrocarbon group derived by the removal of one hydrogen atom from a single carbon atom of a ring system in which two or more identical or non-identical parent aromatic ring systems are joined directly together by a single bond, where the number of such direct ring junctions is one less than the number of parent aromatic ring systems involved. Typical arylaryl groups include, but are not limited to, biphenyl, triphenyl, phenyl-naphthyl, binaphthyl, biphenyl-naphthyl, and the like. Where the number of carbon atoms in an arylaryl group are specified, the numbers refer to the carbon atoms comprising each parent aromatic ring. For example, (C5-C15) arylaryl is an arylaryl group in which each aromatic ring comprises from 5 to 15 carbons, e.g., biphenyl, triphenyl, binaphthyl, phenylnaphthyl, etc. Preferably, each parent aromatic ring system of an arylaryl group is independently a (C5-C15) aromatic, more preferably a (C5-C10) aromatic. Also preferred are arylaryl groups in which all of the parent aromatic ring systems are identical, e.g., biphenyl, triphenyl, binaphthyl, trinaphthyl, etc.

“Biaryl” by itself or as part of another substituent refers to an arylaryl group having two identical parent aromatic systems joined directly together by a single bond. Typical biaryl groups include, but are not limited to, biphenyl, binaphthyl, bianthracyl, and the like. Preferably, the aromatic ring systems are (C5-C15) aromatic rings, more preferably (C5-C10) aromatic rings. A particularly preferred biaryl group is biphenyl.

“Arylalkyl” by itself or as part of another substituent refers to an acyclic alkyl group in which one of the hydrogen atoms bonded to a carbon atom, typically a terminal or sp³ carbon atom, is replaced with an aryl group. Typical arylalkyl groups include, but are not limited to, benzyl, 2-phenylethan-1-yl, 2-phenylethen-1-yl, naphthylmethyl, 2-naphthylethan-1-yl, 2-naphthylethen-1-yl, naphthobenzyl, 2-naphthophenylethan-1-yl and the like. Where specific alkyl moieties are intended, the nomenclature arylalkanyl, arylalkenyl and/or arylalkynyl is used. In preferred embodiments, the arylalkyl group is (C6-C21) arylalkyl, e.g., the alkanyl, alkenyl or alkynyl moiety of the arylalkyl group is (C1-C6) and the aryl moiety is (C5-C15). In particularly preferred embodiments the arylalkyl group is (C6-C13), e.g., the alkanyl, alkenyl or alkynyl moiety of the arylalkyl group is (C1-C3) and the aryl moiety is (C5-C10).

“Parent Heteroaromatic Ring System” refers to a parent aromatic ring system in which one or more carbon atoms are each independently replaced with the same or different heteroatoms or heteroatomic groups. Typical heteroatoms or heteroatomic groups to replace the carbon atoms include, but are not limited to, N, NH, P, O, S, S(O), S(O)₂, Si, etc. Specifically included within the definition of “parent heteroaromatic ring systems” are fused ring systems in which one or more of the rings are aromatic and one or more of the rings are saturated or unsaturated, such as, for example, benzodioxan, benzofuran, chromane, chromene, indole, indoline, xanthene, etc. Also included in the definition of “parent heteroaromatic ring system” are those recognized rings that include common substituents, such as, for example, benzopyrone and 1-methyl-1,2,3,4-tetrazole. Specifically excluded from the definition of “parent heteroaromatic ring system” are benzene rings fused to cyclic polyalkylene glycols such as cyclic polyethylene glycols. Typical parent heteroaromatic ring systems include, but are not limited to, acridine, benzimidazole, benzisoxazole, benzodioxan, benzodioxole, benzofuran, benzopyrone, benzothiadiazole, benzothiazole, benzotriazole, benzoxaxine, benzoxazole, benzoxazoline, carbazole, β-carboline, chromane, chromene, cinnoline, furan, imidazole, indazole, indole, indoline, indolizine, isobenzofuran, isochromene, isoindole, isoindoline, isoquinoline, isothiazole, isoxazole, naphthyridine, oxadiazole, oxazole, perimidine, phenanthridine, phenanthroline, phenazine, phthalazine, pteridine, purine, pyran, pyrazine, pyrazole, pyridazine, pyridine, pyrimidine, pyrrole, pyrrolizine, quinazoline, quinoline, quinolizine, quinoxaline, tetrazole, thiadiazole, thiazole, thiophene, triazole, xanthene, and the like.

“Heteroaryl” by itself or as part of another substituent refers to a monovalent heteroaromatic group having the stated number of ring atoms (e.g., “5-14 membered” means from 5 to 14 ring atoms) derived by the removal of one hydrogen atom from a single atom of a parent heteroaromatic ring system. Typical heteroaryl groups include, but are not limited to, groups derived from acridine, benzimidazole, benzisoxazole, benzodioxan, benzodiaxole, benzofuran, benzopyrone, benzothiadiazole, benzothiazole, benzotriazole, benzoxazine, benzoxazole, benzoxazoline, carbazole, β-carboline, chromane, chromene, cinnoline, furan, imidazole, indazole, indole, indoline, indolizine, isobenzofuran, isochromene, isoindole, isoindoline, isoquinoline, isothiazole, isoxazole, naphthyridine, oxadiazole, oxazole, perimidine, phenanthridine, phenanthroline, phenazine, phthalazine, pteridine, purine, pyran, pyrazine, pyrazole, pyridazine, pyridine, pyrimidine, pyrrole, pyrrolizine, quinazoline, quinoline, quinolizine, quinoxaline, tetrazole, thiadiazole, thiazole, thiophene, triazole, xanthene, and the like, as well as the various hydro isomers thereof. In preferred embodiments, the heteroaryl group is a 5-14 membered heteroaryl, with 5-10 membered heteroaryl being particularly preferred.

“Heteroaryl-Heteroaryl” by itself or as part of another substituent refers to a monovalent heteroaromatic group derived by the removal of one hydrogen atom from a single atom of a ring system in which two or more identical or non-identical parent heteroaromatic ring systems are joined directly together by a single bond, where the number of such direct ring junctions is one less than the number of parent heteroaromatic ring systems involved. Typical heteroaryl-heteroaryl groups include, but are not limited to, bipyridyl, tripyridyl, pyridylpurinyl, bipurinyl, etc. Where the number of atoms are specified, the numbers refer to the number of atoms comprising each parent heteroaromatic ring systems. For example, 5-15 membered heteroaryl-heteroaryl is a heteroaryl-heteroaryl group in which each parent heteroaromatic ring system comprises from 5 to 15 atoms, e.g., bipyridyl, tripyridyl, etc. Preferably, each parent heteroaromatic ring system is independently a 5-15 membered heteroaromatic, more preferably a 5-10 membered heteroaromatic. Also preferred are heteroaryl-heteroaryl groups in which all of the parent heteroaromatic ring systems are identical.

“Biheteroaryl” by itself or as part of another substituent refers to a heteroaryl-heteroaryl group having two identical parent heteroaromatic ring systems joined directly together by a single bond. Typical biheteroaryl groups include, but are not limited to, bipyridyl, bipurinyl, biquinolinyl, and the like. Preferably, the heteroaromatic ring systems are 5-15 membered heteroaromatic rings, more preferably 5-10 membered heteroaromatic rings.

“Heteroarylalkyl” by itself or as part of another substituent refers to an acyclic alkyl group in which one of the hydrogen atoms bonded to a carbon atom, typically a terminal or sp³ carbon atom, is replaced with a heteroaryl group. Where specific alkyl moieties are intended, the nomenclature heteroarylalkanyl, heteroarylalkenyl and/or heteroarylalkynyl is used. In preferred embodiments, the heteroarylalkyl group is a 6-21 membered heteroarylalkyl, e.g., the alkanyl, alkenyl or alkynyl moiety of the heteroarylalkyl is (C1-C6) alkyl and the heteroaryl moiety is a 5-15-membered heteroaryl. In particularly preferred embodiments, the heteroarylalkyl is a 6-13 membered heteroarylalkyl, e.g., the alkanyl, alkenyl or alkynyl moiety is (C1-C3) alkyl and the heteroaryl moiety is a 5-10 membered heteroaryl.

“Halogen” or “Halo” by themselves or as part of another substituent, unless otherwise stated, refer to fluoro, chloro, bromo and iodo.

“Haloalkyl” by itself or as part of another substituent refers to an alkyl group in which one or more of the hydrogen atoms is replaced with a halogen. Thus, the term “haloalkyl” is meant to include monohaloalkyls, dihaloalkyls, trihaloalkyls, etc. up to perhaloalkyls. For example, the expression “(C1-C2) haloalkyl” includes fluoromethyl, difluoromethyl, trifluoromethyl, 1-fluoroethyl, 1,1-difluoroethyl, 1,2-difluoroethyl, 1,1,1-trifluoroethyl, perfluoroethyl, etc.

The above-defined groups may include prefixes and/or suffixes that are commonly used in the art to create additional well-recognized substituent groups. As examples, “alkyloxy” or “alkoxy” refers to a group of the formula —OR″, “alkylamine” refers to a group of the formula —NHR″ and “dialkylamine” refers to a group of the formula —NR″R″, where each R″ is independently an alkyl. As another example, “haloalkoxy” or “haloalkyloxy” refers to a group of the formula —OR′″, where R′″ is a haloalkyl.

“Protecting group” refers to a group of atoms that, when attached to a reactive functional group in a molecule, mask, reduce or prevent the reactivity of the functional group. Typically, a protecting group may be selectively removed as desired during the course of a synthesis. Examples of protecting groups can be found in Greene and Wuts, Protective Groups in Organic Chemistry, 3^(rd) Ed., 1999, John Wiley & Sons, NY and Harrison et al., Compendium of Synthetic Organic Methods, Vols. 1-8, 1971-1996, John Wiley & Sons, NY. Representative amino protecting groups include, but are not limited to, formyl, acetyl, trifluoroacetyl, benzyl, benzyloxycarbonyl (“CBZ”), tert-butoxycarbonyl (“Boc”), trimethylsilyl (“TMS”), 2-trimethylsilyl-ethanesulfonyl (“TES”), trityl and substituted trityl groups, allyloxycarbonyl, 9-fluorenylmethyloxycarbonyl (“FMOC”), nitro-veratryloxycarbonyl (“NVOC”) and the like. Representative hydroxyl protecting groups include, but are not limited to, those where the hydroxyl group is either acylated or alkylated such as benzyl and trityl ethers, as well as alkyl ethers, tetrahydropyranyl ethers, trialkylsilyl ethers (e.g., TMS or TIPPS groups) and allyl ethers.

“Prodrug” refers to a derivative of an active 2,4-pyrimidinediamine compound (drug) that requires a transformation under the conditions of use, such as within the body, to release the active 2,4-pyrimidinediamine drug. Prodrugs are frequently, but not necessarily, pharmacologically inactive until converted into the active drug. Prodrugs are typically obtained by masking a functional group in the 2,4-pyrimidinediamine drug believed to be in part required for activity with a progroup (defined below) to form a promoiety which undergoes a transformation, such as cleavage, under the specified conditions of use to release the functional group, and hence the active 2,4-pyrimidinediamine drug. The cleavage of the promoiety may proceed spontaneously, such as by way of a hydrolysis reaction, or it may be catalyzed or induced by another agent, such as by an enzyme, by light, by acid or base, or by a change of or exposure to a physical or environmental parameter, such as a change of temperature. The agent may be endogenous to the conditions of use, such as an enzyme present in the cells to which the prodrug is administered or the acidic conditions of the stomach, or it may be supplied exogenously.

A wide variety of progroups, as well as the resultant promoieties, suitable for masking functional groups in the active 2,4-pyrimidinediamines compounds to yield prodrugs are well-known in the art. For example, a hydroxyl functional group may be masked as a sulfonate, ester or carbonate promoiety, which may be hydrolyzed in vivo to provide the hydroxyl group. An amino functional group may be masked as an amide, carbamate, imine, urea, phosphenyl, phosphoryl or sulfenyl promoiety, which may be hydrolyzed in vivo to provide the amino group. A carboxyl group may be masked as an ester (including silyl esters and thioesters), amide or hydrazide promoiety, which may be hydrolyzed in vivo to provide the carboxyl group. Other specific examples of suitable progroups and their respective promoieties will be apparent to those of skill in the art.

“Progroup” refers to a type of protecting group that, when used to mask a functional group within an active 2,4-pyrimidinediamine drug to form a promoiety, converts the drug into a prodrug. Progroups are typically attached to the functional group of the drug via bonds that are cleavable under specified conditions of use. Thus, a progroup is that portion of a promoiety that cleaves to release the functional group under the specified conditions of use. As a specific example, an amide promoiety of the formula —NH—C(O)CH₃ comprises the progroup —C(O)CH₃.

“Fc Receptor” refers to a member of the family of cell surface molecules that binds the Fc portion (containing the specific constant region) of an immunoglobulin. Each Fc receptor binds immunoglobulins of a specific type. For example the Fcα receptor (“FcαR”) binds IgA, the FcεR binds IgE and the FcγR binds IgG.

The FcαR family includes the polymeric Ig receptor involved in epithelial transport of IgA/IgM, the mycloid specific receptor RcαRI (also called CD89), the Fcα/μR and at least two alternative IgA receptors (for a recent review see Monteiro & van de Winkel, 2003, Annu. Rev. Immunol, advanced e-publication. The FcαRI is expressed on neutrophils, eosinophils, monocytes/macrophages, dendritic cells and kupfer cells. The FcγRI includes one alpha chain and the FcR gamma homodimer that bears an activation motif (ITAM) in the cytoplasmic domain and phosphorylates Syk kinase.

The FcεR family includes two types, designated FcεRI and FcεRII (also known as CD23). FcεRI is a high affinity receptor (binds IgE with an affinity of about 10¹⁰ M⁻¹) found on mast, basophil and eosinophil cells that anchors monomeric IgE to the cell surface. The FcεRI possesses one alpha chain, one beta chain and the gamma chain homodimer discussed above. The FcεRII is a low affinity receptor expressed on mononuclear phagocytes, B lymphocytes, eosinophils and platelets. The FcεRII comprises a single polypeptide chain and does not include the gamma chain homodimer.

The FcγR family includes three types, designated FcγRI (also known as CD64), FcγRII (also known as CD32) and FcγRIII (also known as CD16). FcγRI is a high affinity receptor (binds IgG1 with an affinity of 10⁸ M⁻¹) found on mast, basophil, mononuclear, neutrophil, eosinophil, deudritic and phagocyte cells that anchors nomomeric IgG to the cell surface. The FcγRI includes one alpha chain and the gamma chain dimer shared by FcγRI and FcεRI.

The FcγRII is a low affinity receptor expressed on neutrophils, monocytes, eosinophils, platelets and B lymphocytes. The FcγRII includes one alpha chain, and does not include the gamma chain homodimer discussed above.

The FcγRIII is a low affinity (binds IgG1 with an affinity of 5×10⁵ M⁻¹) expressed on NK, eosinophil, macrophage, neutrophil and mast cells. It comprises one alpha chain and the gamma homodimer shared by FcαRI, FcεRI and FcγRI.

Skilled artisans will recognize that the subunit structure and binding properties of these various Fc receptors, cell types expressing them, are not completely characterized.

The above discussion merely reflects the current state-of-the-art regarding these receptors (see, e.g., Immunobiology: The Immune System in Health & Disease, 5^(th) Edition, Janeway et al., Eds, 2001, ISBN 0-8153-3642-x, FIG. 9.30 at pp. 371), and is not intended to be limiting with respect to the myriad receptor signaling cascades that can be regulated with the compounds described herein.

“Fc Receptor-Mediated Degranulation” or “Fc Receptor-Induced Degranulation” refers to degranulation that proceeds via an Fc receptor signal transduction cascade initiated by crosslinking of an Fc receptor.

“IgE-Induced Degranulation” or “FcεRI-Mediated Degranulation” refers to degranulation that proceeds via the IgE receptor signal transduction cascade initiated by crosslinking of FcεR1-bound IgE. The crosslinking may be induced by an IgE-specific allergen or other multivalent binding agent, such as an anti-IgE antibody. Referring to FIG. 2, in mast and/or basophil cells, the FcεRI signaling cascade leading to degranulation may be broken into two stages: upstream and downstream. The upstream stage includes all of the processes that occur prior to calcium ion mobilization (illustrated as “Ca²+” in FIG. 2; see also FIG. 3). The downstream stage includes calcium ion mobilization and all processes downstream thereof. Compounds that inhibit FcεRI-mediated degranulation may act at any point along the FcεRI-mediated signal transduction cascade. Compounds that selectively inhibit upstream FcεRI-mediated degranulation act to inhibit that portion of the FcεRI signaling cascade upstream of the point at which calcium ion mobilization is induced. In cell-based assays, compounds that selectively inhibit upstream FcεRI-mediated degranulation inhibit degranulation of cells such as mast or basophil cells that are activated or stimulated with an IgE-specific allergen or binding agent (such as an anti-IgE antibody) but do not appreciably inhibit degranulation of cells that are activated or stimulated with degranulating agents that bypass the FcεRI signaling pathway, such as, for example the calcium ionophores ionomycin and A23187.

“IgG-Induced Degranulation” or “FcγRI-Mediated Degranulation” refers to degranulation that proceeds via the FcγRI signal transduction cascade initiated by crosslinking of FcγRI-bound IgG. The crosslinking may be induced by an IgG-specific allergen or another multivalent binding agent, such as an anti-IgG or fragment antibody. Like the FcεRI signaling cascade, in mast and basophil cells the FcγRI signaling cascade also leads to degranulation which may be broken into the same two stages: upstream and downstream. Similar to FcεRI-mediated degranulation, compounds that selectively inhibit upstream FcγRI-mediated degranulation act upstream of the point at which calcium ion mobilization is induced. In cell-based assays, compounds that selectively inhibit upstream FcγRI-mediated degranulation inhibit degranulation of cells such as mast or basophil cells that are activated or stimulated with an IgG-specific allergen or binding agent (such as an anti-IgG antibody or fragment) but do not appreciably inhibit degranulation of cells that are activated or stimulated with degranulating agents that bypass the FcγRI signaling pathway, such as, for example the calcium ionophores ionomycin and A23187.

“Ionophore-Induced Degranulation” or “Ionophore-Mediated Degranulation” refers to degranulation of a cell, such as a mast or basophil cell, that occurs upon exposure to a calcium ionophore such as, for example, ionomycin or A23187.

“Syk Kinsase” refers to the well-known 72 kDa non-receptor (cytoplasmic) spleen protein tyrosine kinase expressed in B-cells and other hematopoetic cells. Syk kinase includes two consensus Src-homology 2 (SH2) domains in tandem that bind to phosphorylated immunoreceptor tyrosine-based activation motifs (“ITAMs”), a “linker” domain and a catalytic domain (for a review of the structure and function of Syk kinase see Sada et al., 2001, J. Biochem. (Tokyo) 130:177-186); see also Turner et al., 2000, Immunology Today 21:148-154). Syk kinase has been extensively studied as an effector of B-cell receptor (BCR) signaling (Turner et al., 2000, supra). Syk kinase is also critical for tyrosine phosphorylation of multiple proteins which regulate important pathways leading from immunoreceptors, such as Ca²⁺ mobilization and mitogen-activated protein kinase (MAPK) cascades (see, e.g., FIG. 2) and degranulation. Syk kinase also plays a critical role in integrin signaling in neutrophils (see, e.g., Mocsai et al. 2002, Immunity 16:547-558).

As used herein, Syk kinase includes kinases from any species of animal, including but not limited to, homosapiens, simian, bovine, porcine, rodent, etc., recognized as belonging to the Syk family. Specifically included are isoforms, splice variants, allelic variants, mutants, both naturally occurring and man-made. The amino acid sequences of such Syk kinases are well known and available from GENBANK. Specific examples of mRNAs encoding different isoforms of human Syk kinase can be found at GENBANK accession no. gi|21361552|ref|NM_(—)003177.2|, gi|496899|emb|Z29630.1|HSSYKPTK[496899] and gi|15030258|gb|BC011399.1|BC011399[15030258], which are incorporated herein by reference.

Skilled artisans will appreciate that tyrosine kinases belonging to other families may have active sites or binding pockets that are similar in three-dimensional structure to that of Syk. As a consequence of this structural similarity, such kinases, referred to herein as “Syk mimics,” are expected to catalyze phosphorylation of substrates phosphorylated by Syk. Thus, it will be appreciated that such Syk mimics, signal transduction cascades in which such Syk mimics play a role and biological responses effected by such Syk mimics and Syk mimic-dependent signaling cascades may be regulated, and in particular inhibited, with the 2,4-pyrimidinediamine compounds described herein.

“Syk-Dependent Signaling Cascade” refers to a signal transduction cascade in which Syk kinase plays a role. Non-limiting examples of such Syk-dependent signaling cascades include the FcαRI, FcεRI, FcγRI, FcγRIII, BCR and integrin signaling cascades.

“Autoimmune Disease” refers to those diseases which are commonly associated with the nonanaphylactic hypersensitivity reactions (Type II, Type III and/or Type IV hypersensitivity reactions) that generally result as a consequence of the subject's own humoral and/or cell-mediated immune response to one or more immunogenic substances of endogenous and/or exogenous origin. Such autoimmune diseases are distinguished from diseases associated with the anaphylactic (Type I or IgE-mediated) hypersensitivity reactions.

6.2 The 2,4-Pyrimidinediamine Compounds

The compounds of the invention are generally 2,4-pyrimidinediamine compounds according to structural formula (I):

including salts, hydrates, solvates and N-oxides thereof, wherein:

L¹ and L² are each, independently of one another, selected from the group consisting of a direct bond and a linker;

R² is selected from the group consisting of (C1-C6) alkyl optionally substituted with one or more of the same or different R⁸ groups, (C3-C8) cycloalkyl optionally substituted with one or more of the same or different R⁸ groups, cyclohexyl optionally substituted with one or more of the same or different R⁸ groups, 3-8 membered cycloheteroalkyl optionally substituted with one or more of the same or different R⁸ groups, (C5-C15) aryl optionally substituted with one or more of the same or different R⁸ groups, phenyl optionally substituted with one or more of the same or different R⁸ groups and 5-15 membered heteroaryl optionally substituted with one or more of the same or different R⁸ groups;

R⁴ is selected from the group consisting of hydrogen, (C1-C6) alkyl optionally substituted with one or more of the same or different R⁸ groups, (C3-C8) cycloalkyl optionally substituted with one or more of the same or different R⁸ groups, cyclohexyl optionally substituted with one or more of the same or different R⁸ groups, 3-8 membered cycloheteroalkyl optionally substituted with one or more of the same or different R⁸ groups, (C5-C15) aryl optionally substituted with one or more of the same or different R⁸ groups, phenyl optionally substituted with one or more of the same or different R⁸ groups and 5-15 membered heteroaryl optionally substituted with one or more of the same or different R⁸ groups;

R⁵ is selected from the group consisting of R⁶, (C1-C6) alkyl optionally substituted with one or more of the same or different R⁸ groups, (C1-C4) alkanyl optionally substituted with one or more of the same or different R⁸ groups, (C2-C4) alkenyl optionally substituted with one or more of the same or different R⁸ groups and (C2-C4) alkynyl optionally substituted with one or more of the same or different R⁸ groups;

each R⁶ is independently selected from the group consisting of hydrogen, an electronegative group, —OR^(d), —SR^(d), (C1-C3) haloalkyloxy, (C1-C3) perhaloalkyloxy, —NR^(c)R^(c), halogen, (C1-C3) haloalkyl,(C1-C3) perhaloalkyl, —CF₃, —CH₂CF₃, —CF₂CF₃, —CN, —NC, —OCN, —SCN, —NO, —NO₂, —N₃, —S(O)R^(d), —S(O)₂R^(d), —S(O)₂OR^(d), —S(O)NR^(c)R^(c), —S(O)₂NR^(c)R^(c), —OS(O)R^(d), —OS(O)₂R^(d), —OS(O)₂OR^(d), —OS(O)NR^(c)R^(c), —OS(O)₂NR^(c)R^(c), —C(O)R^(d), —C(O)OR^(d), —C(O)NR^(c)R^(c), —C(NH)NR^(c)R^(c), —OC(O)R^(d), —SC(O)R^(d), —OC(O)OR^(d), —SC(O)OR^(d), —OC(O)NR^(c)R^(c), —SC(O)NR^(c)R^(c), —OC(NH)NR^(c)R^(c), —SC(NH)NR^(c)R^(c), —[NHC(O)]_(n)R^(d), —[NHC(O)]_(n)OR^(d), —[NHC(O)]_(n)NR^(c)R^(c) and -[NHC(NH)]_(n)NR^(c)R^(c), (C5-C10) aryl optionally substituted with one or more of the same or different R⁸ groups, phenyl optionally substituted with one or more of the same or different R⁸ groups, (C6-C16) arylalkyl optionally substituted with one or more of the same or different R⁸ groups, 5-10 membered heteroaryl optionally substituted with one or more of the same or different R⁸ groups and 6-16 membered heteroarylalkyl optionally substituted with one or more of the same or different R⁸ groups;

R⁸ is selected from the group consisting of R^(a), R^(b), R^(a) substituted with one or more of the same or different R^(a) or R^(b), —OR^(a) substituted with one or more of the same or different R^(a) or R^(b), —B(OR^(a))₂, —B(NR^(c)R^(c))₂, —(CH₂)_(m)—R^(b), —(CHR^(a))_(m)—R^(b), —O—(CH₂)_(m)—R^(b), —S—(CH₂)_(m)—R^(b), —O—CHR^(a)R^(b), —O—CR^(a)(R^(b))₂, —O—(CHR^(a))_(m)—R^(b), —O— (CH₂)_(m)—CH[(CH₂)_(m)R^(b)]R^(b), —S—(CHR^(a))_(m)—R^(b), —C(O)NH—(CH₂)_(m)—R^(b), —C(O)NH—(CHR^(a))_(m)—R^(b), —O—(CH₂)_(m)—C(O)NH—(CH₂)_(m)—R^(b), —S—(CH₂)_(m)—C(O)NH—(CH₂)_(m)—R^(b), —O—(CHR^(a))_(m)—C(O)NH—(CHR^(a))_(m)—R^(b), —S—(CHR^(a))_(m)—C(O)NH—(CHR^(a))_(m)—R^(b), —NH—(CH₂)_(m)—R^(b), —NH—(CHR^(a))_(m)—R^(b), —NH[(CH₂)_(m)R^(b)], —N[(CH₂)_(m)R^(b)]₂, —NH—C(O)—NH—(CH₂)_(m)—R^(b), —NH—C(O)—(CH₂)_(m)—CHR^(b)R^(b) and —NH—(CH₂)_(m)—C(O)—NH—(CH₂)_(m)—R^(b);

each R^(a) is independently selected from the group consisting of hydrogen, (C1-C6) alkyl, (C3-C8) cycloalkyl, cyclohexyl, (C4-C11) cycloalkylalkyl, (C5-C10) aryl, phenyl, (C6-C16) arylalkyl, benzyl, 2-6 membered heteroalkyl, 3-8 membered cycloheteroalkyl, morpholinyl, piperazinyl, homopiperazinyl, piperidinyl, 4-11 membered cycloheteroalkylalkyl, 5-10 membered heteroaryl and 6-16 membered heteroarylalkyl;

each R^(b) is a suitable group independently selected from the group consisting of ═O, —OR^(d), (C1-C3) haloalkyloxy, —OCF₃, ═S, —SR^(d), ═NR^(d), ═NOR^(d), —NR^(c)R^(c), halogen, —CF₃, —CN, —NC, —OCN, —SCN, —NO, —NO₂, ═N₂, —N₃, —S(O)R^(d), —S(O)₂R^(d), —S(O)₂OR^(d), —S(O)NR^(c)R^(c), —S(O)₂NR^(c)R^(c), —OS(O)R^(d), —OS(O)₂R^(d), —OS(O)₂OR^(d), —OS(O)₂NR^(c)R^(c), —C(O)R^(d), —C(O)OR^(d), —C(O)NR^(c)R^(c), —C(NH)NR^(c)R^(c), —C(NR^(a))NR^(c)R^(c), —C(NOH)R^(a), —C(NOH)NR^(c)R^(c), —OC(O)R^(d), —OC(O)OR^(d), —OC(O)NR^(c)R^(c), —OC(NH)NR^(c)R^(c), —OC(NR^(a))NR^(c)R^(c), —[NHC(O)]_(n)R^(d), —[NR^(a)C(O)]_(n)R^(d), —[NHC(O)]_(n)OR^(d), —[NR^(a)C(O)]_(n)OR^(d), —[NHC(O)]_(n)NR^(c)R^(c), —[NR^(a)C(O)]_(n)NR^(c)R^(c), —[NHC(NH)]_(n)NR^(c)R^(c) and —[NR^(a)C(NR^(a))]_(n)NR^(c)R^(c);

each R^(c) is independently R^(a), or, alternatively, each R^(c) is taken together with the nitrogen atom to which it is bonded to form a 5 to 8-membered cycloheteroalkyl or heteroaryl which may optionally include one or more of the same or different additional heteroatoms and which is optionally substituted with one or more of the same or different R^(a) or suitable R^(b) groups;

each R^(d) is independently R^(a);

-   -   each m is independently an integer from 1 to 3; and

each n is independently an integer from 0 to 3.

In the compounds of structural formula (I), L¹ and L² represent, independently of one another, a direct bond or a linker. Thus, as will be appreciated by skilled artisans, the substituents R² and/or R⁴ may be bonded either directly to their respective nitrogen atoms or, alternatively, spaced away from their respective nitrogen atoms by way of a linker. The identity of the linker is not critical and typical suitable linkers include, but are not limited to, (C1-C6) alkyldiyls, (C1-C6) alkanos and (C1-C6) heteroalkyldiyls, each of which may be optionally substituted with one or more of the same or different R⁸ groups, where R⁸ is as previously defined for structural formula (I). In a specific embodiment, L¹ and L² are each, independently of one another, selected from the group consisting of a direct bond, (C1-C3) alkyldiyl optionally substituted with one or more of the same or different R^(a), suitable R^(b) or R⁹ groups and 1-3 membered heteroalkyldiyl optionally substituted with one or more of the same or different R^(a), suitable R^(b) or R⁹ groups, wherein R⁹ is selected from the group consisting of (C1-C3) alkyl, —OR^(a), —C(O)OR^(a), (C5-C10) aryl optionally substituted with one or more of the same or different halogens, phenyl optionally substituted with one or more of the same or different halogens, 5-10 membered heteroaryl optionally substituted with one or more of the same or different halogens and 6 membered heteroaryl optionally substituted with one or more of the same or different halogens; and R^(a) and R^(b) are as previously defined for structural formula (I). Specific R⁹ groups that may be used to substitute L¹ and L² include —OR^(a), —C(O)OR^(a), phenyl, halophenyl and 4-halophenyl, wherein R^(a) is as previously defined for structural formula (I).

In another specific embodiment, L¹ and L² are each, independently of one another, selected from the group consisting of methano, ethano and propano, each of which may be optionally monosubstituted with an R⁹ group, where R⁹ is as previously defined above.

In all of the above embodiments, specific R^(a) groups that may be included in R⁹ groups are selected from the group consisting of hydrogen, (C1-C6) alkyl, phenyl and benzyl.

In still another specific embodiment, L¹ and L² are each a direct bond such that the 2,4-pyrimidinediamine compounds of the invention are compounds according to structural formula (Ia):

including salts, hydrates, solvates and N-oxides thereof, wherein R², R⁴, R⁵ and R⁶ are as previously defined for structural formula (I). Additional specific embodiments of the 2,4-pyrimidinediamine compounds of the invention are described below.

In a first embodiment of the compounds of structural formulae (I) and (Ia), R², R⁴, R⁵, R⁶, L¹ and L² are as previously defined for their respective structures (I) and (Ia), with the proviso that R² is not 3,4,5-trimethoxyphenyl, 3,4,5-tri (C1-C6) alkoxyphenyl or

where R²¹, R²² and R²³ are as defined for R¹, R² and R³, respectively of U.S. Pat. No. 6,235,746, the disclosure of which is incorporated by reference. In a specific embodiment of this first embodiment, R²¹ is hydrogen, halo, straight-chain or branched (C1-C6) alkyl optionally substituted with one or more of the same or different R²⁵ groups, hydroxyl, (C1-C6) alkoxy optionally substituted with one or more of the same or different phenyl or R²⁵ groups, thiol (—SH), (C1-C6) alkylthio optionally substituted with one or more of the same or different phenyl or R²⁵ groups, amino (—NH₂), —NHR²⁶ or —NR²⁶R²⁶; R²² and R²³ are each, independently of one another, a (C1-C6) straight-chain or branched alkyl optionally substituted with one or more of the same or different R²⁵ groups; R²⁵ is selected from the group consisting of halo, hydroxyl, (C1-C6) alkoxy, thiol, (C1-C6) alkylthio, (C1-C6) alkylamino and (C1-C6) dialkylamino; and each R²⁶ is independently a (C1-C6) alkyl optionally substituted with one or more of the same or different phenyl or R²⁵ groups or a —C(O)R²⁷, where R²⁷ is a (C1-C6) alkyl optionally substituted with one or more of the same or different phenyl or R²⁵ groups.

In another specific embodiment of this first embodiment, R²¹ is methoxy optionally substituted with one or more of the same or different halo groups and/or R²² and R²³ are each, independently of one another, a methyl or ethyl optionally substituted with one or more of the same or different halo groups.

In a second embodiment of the compounds of structural formulae (I) and (Ia), R², R⁴, R⁵ and L² are as previously described for their respective structures (I) and (Ia), L¹ is a direct bond and R⁶ is hydrogen, with the proviso that R² is not 3,4,5-trimethoxyphenyl, 3,4,5-tri (C1-C6) alkoxyphenyl or

where R²¹, R²² and R²³ are as defined above, in connection with the first embodiment.

In a third embodiment, the 2,4-pyrimidinediamine compounds of structural formulae (I) and (Ia) exclude one or more of the following compounds:

-   N2,N4-bis(4-ethoxyphenyl)-5-fluoro-2,4-pyrimidinediamine (R070790); -   N2,N4-bis(2-methoxyphenyl)-5-fluoro-2,4-pyrimidinediamine (R081166); -   N2,N4-bis(4-methoxyphenyl)-5-fluoro-2,4-pyrimidinediamine (R088814); -   N2,N4-bis(2-chlorophenyl)-5-fluoro-2,4-pyrimidinediamine (R088815); -   N2,N4-bisphenyl-5-fluoro-2,4-pyrimidinediamine (R091880); -   N2,N4-bis(3-methylphenyl)-5-fluoro-2,4-pyrimidinediamine (R092788); -   N2,N4-bis(3-chlorophenyl)-5-fluoro-2,4-pyrimidinediamine (R067962); -   N2,N4-bis(2,5-dimethylphenyl)-5-fluoro-2,4-pyrimidinediamine     (R067963); -   N2,N4-bis(3,4-dimethylphenyl)-5-fluoro-2,4-pyrimidinediamine     (R067964); -   N2,N4-bis(4-chlorophenyl)-5-fluoro-2,4-pyrimidinediamine (R0707153); -   N2,N4-bis(2,4-dimethylphenyl)-5-fluoro-2,4-pyrimidinediamine     (R070791); -   N2,N4-bis(3-bromophenyl)-5-fluoro-2,4-pyrimidinediamine (R008958); -   N2,N4-bis(phenyl)-5-fluoro-2,4-pyrimidinediamine; -   N2,N4-bis(morpholino)-5-fluoro-2,4-pyrimidinediamine; and -   N2,N4-bis[(3-chloro-4-methoxyphenyl)]-5-fluoro-2,4-pyrimidinediamine

In a fourth embodiment, the compounds of structural formulae (I) and (Ia) exclude compounds according to the following structural formula (Ib):

wherein R²⁴ is (C1-C6) alkyl; and R²¹, R²² and R²³ are as previously defined in connection with the first embodiment.

In a fifth embodiment, the compounds of structural formulae (I) and (Ia) exclude the compounds described in Examples 1-141 of U.S. Pat. No. 6,235,746, the disclosure of which is incorporated herein by reference.

In a sixth embodiment, the compounds of structural formulae (I) and (Ia) exclude compounds defined by formula (I) or formula I(a) of this U.S. Pat. No. 6,235,746 (see, e.g., the disclosure at Col. 1, line 48 through Col. 7, line 49 and Col. 8, lines 9-36, which is incorporated by reference).

In a seventh embodiment, the compounds of structural formulae (I) and (Ia) exclude compounds in which R⁵ is cyano or —C(O)NHR, where R is hydrogen or (C1-C6) alkyl, when R² is a substituted phenyl; R⁴ is a substituted or unsubstituted (C1-C6) alkyl, (C3-C8) cycloalkyl, 3-8 membered cycloheteroalkyl or 5-15 membered heteroaryl; and R⁶ is hydrogen.

In an eighth embodiment, the compounds of structural formulae (I) and (Ia) exclude the compounds defined by formulae (I) and (X) of WO 02/04429 or any compound disclosed in WO 02/04429, the disclosure of which is incorporated herein by reference.

In a ninth embodiment of the compounds of structural formulae (I) and (Ia), when R⁵ is cyano or —C(O)NHR, where R is hydrogen or (C1-C6) alkyl; and R⁶ is hydrogen, then R² is other than a substituted phenyl group.

In a tenth embodiment, the compounds of structural formulae (I) and (Ia) exclude compounds in which R² and R⁴ are each independently a substituted or unsubstituted pyrrole or indole ring which is attached to the remainder of the molecule via its ring nitrogen atom.

In an eleventh embodiment, the compounds of structural formulae (I) and (Ia) exclude compounds defined by formulae (I) and (IV) of U.S. Pat. No. 4,983,608 or any compound disclosed in U.S. Pat. No. 4,983,608, the disclosure of which is incorporated herein by reference.

Those of skill in the art will appreciate that in the compounds of formulae (I) and (Ia), R² and R⁴ may be the same or different, and may vary broadly. When R² and/or R⁴ are optionally substituted rings, such as optionally substituted cycloalkyls, cycloheteroalkyls, aryls and heteroaryls, the ring may be attached to the remainder of the molecule through any available carbon or heteroatom. The optional substituents may be attached to any available carbon atoms and/or heteroatoms.

In a twelfth embodiment of the compounds of structural formulae (I) and (Ia), R² and/or R⁴ is an optionally substituted phenyl or an optionally substituted (C5-C15) aryl, subject to the provisos that (1) when R⁶ is hydrogen, then R² is not 3,4,5-trimethoxyphenyl or 3,4,5-tri (C1-C6) alkoxyphenyl; (2) when R² is a 3,4,5-trisubstituted phenyl, then the substituents at the 3- and 4-positions are not simultaneously methoxy or (C1-C6) alkoxy; or (3) when R⁶ is hydrogen and R⁴ is (C1-C6) alkyl, (C₃-C₈) cycloalkyl, 3-8 membered cycloheteroalkyl or 5-15 membered heteroaryl, then R⁵ is other than cyano. Alternatively, R² is subject to the provisos described in connection with the first or second embodiments. The optionally substituted aryl or phenyl group may be attached to the remainder of the molecule through any available carbon atom. Specific examples of optionally substituted phenyls include phenyls that are optionally mono-, di- or tri-substituted with the same or different R⁸ groups, where R⁸ is as previously defined for structural formula (I) and subject to the above provisos. When the phenyl is mono-substituted, the R⁸ substituent may be positioned at either the ortho, meta or para position. When positioned at the ortho, meta or para position, R⁸ is preferably selected from the group consisting of (C1- C10) alkyl, (C1-C10) branched alkyl, —OR^(a) optionally substituted with one or more of the same or different R^(b) groups, —O—C(O)OR^(a), —O—(CH₂)_(m)—C(O)OR^(a), —C(O)OR^(a), —O—(CH₂)_(m)—NR^(c)R^(c), —O—C(O)NR^(c)R^(c), —O—(CH₂)_(m)—C(O)NR^(c)R^(c), —O—C(NH)NR^(c)R^(c), —O—(CH₂)_(m)—C(NH)NR^(c)R^(c) and —NH—(CH₂)_(m)—NR^(c)R^(c), where m, R^(a) and R^(c) are as previously defined for structural formula (I). In one embodiment of these compounds, —NR^(c)R^(c) is a 5-6 membered heteroaryl which optionally includes one or more of the same or different additional heteroatoms. Specific examples of such 5-6 membered heteroaryls include, but are not limited to, oxadiazolyl, triazolyl, thiazolyl, oxazolyl, tetrazolyl and isoxazolyl.

In another embodiment of these compounds, —NR^(c)R^(c) is a 5-6 membered saturated cycloheteroalkyl ring which optionally includes one or more of the same or different heteroatoms. Specific examples of such cycloheteroalkyls include, but are not limited to, pyrrolidinyl, pyrazolidinyl, imidazolidinyl, piperidinyl, piperazinyl and morpholinyl.

In still another embodiment of these compounds, each R^(a) is independently a (C1-C6) alkyl and/or each —NR^(c)R^(c) is —NHR^(a), where R^(a) is a (C1-C6) alkyl. In one specific embodiment, R⁸ is —O—CH₂—C(O)NHCH₃. In another specific embodiment R⁸ is —OH.

When the phenyl is di-substituted or tri-substituted, the R⁸ substituents may be positioned at any combination of positions. For example, the R⁸ substituents may be positioned at the 2,3-, 2,4-, 2,5-, 2,6-, 3,4-, 3,5-, 2,3,4-, 2,3,5-, 2,3,6-, 2,4,5-, 2,4,6-, 2,5,6- or 3,4,5-positions. In one embodiment of compounds including a disubstituted phenyl, the substituents are positioned other than 3,4. In another embodiment they are positioned 3,4. In one embodiment of compounds including a trisubstituted phenyl, the substituents are positioned other than 3,4,5 or, alternatively, no two of the substituents are positioned 3,4. In another embodiment, the substituents are positioned 3,4,5.

Specific examples of R⁸ substituents in such di- and trisubstituted phenyls include the various R⁸ substituents described above in connection with the ortho, meta and para substituted phenyls.

In another specific embodiment, R⁸ substituents useful for substituting such di- and trisubstituted phenyls include (C1-C6) alkyl, (C1-C6) alkoxy, methoxy, halo, chloro, (C1-C6) perhaloalkyl, —CF₃, (C1-C6) perhaloalkoxy and —OCF₃. In a preferred embodiment, such R⁸ substituents are positioned 3, 4 or 3,5. Specific examples of preferred di-substituted phenyl rings include 3-chloro-4-methoxy-phenyl, 3-methoxy-4-chlorophenyl, 3-chloro-4-trifluoromethoxy-phenyl, 3-trifluoromethoxy-4-chloro-phenyl, 3,4-dichloro-phenyl, 3,4-dimethoxyphenyl and 3,5-dimethoxyphenyl, with the provisos that: (1) when R⁴ is one of the above-identified phenyls, and R⁵ and R⁶ are each hydrogen, then R² is not 3,4,5-tri(C1-C6)alkoxyphenyl or 3,4,5-trimethoxyphenyl; (2) when R² is 3,4-dimethoxyphenyl and R⁵ and R⁶ are each hydrogen, then R⁴ is not 3-(C1-C6)alkoxyphenyl, 3-methoxyphenyl, 3,4-di-(C1-C6) alkoxyphenyl or 3,4-dimethoxyphenyl; (3) when R⁴ is 3-chloro-4-methoxyphenyl and R⁵ is halo or fluoro, and optionally R⁶ is hydrogen, then R² is not 3-chloro-4-(C1-C6)alkoxyphenyl or 3-chloro-4-methoxyphenyl; (4) when R⁴ is 3,4-dichlorophenyl, R⁵ is hydrogen, (C1-C6) alkyl, methyl, halo or chloro and optionally R⁶ is hydrogen, then R² is not a phenyl mono substituted at the para position with a (C1-C6) alkoxy group which is optionally substituted with one or more of the same or different R^(b), —OH or —NR^(c)R^(c) groups, where R^(b) and R^(c) are as previously described for structural formula (I); and/or (5) R² and/or R⁴ is not 3,4,5-tri(C1-C6)alkoxyphenyl or 3,4,5-trimethoxyphenyl, especially when R⁵ and R⁶ are each hydrogen.

In another embodiment of compounds including a trisubstituted phenyl, the trisubstituted phenyl has the formula:

wherein: R³¹ is methyl or (C1-C6) alkyl; R³² is hydrogen, methyl or (C1-C6) alkyl; and R³³ is a halo group.

In a thirteenth embodiment of the compounds of structural formulae (I) and (Ia), R² and/or R⁴ is an optionally substituted heteroaryl. Typical heteroaryl groups according to this thirteenth embodiment comprise from 5 to 15, and more typically from 5 to 11 ring atoms, and include one, two, three or four of the same or different heteroatoms or heteroatomic groups selected from the group consisting of N, NH, O, S, S(O) and S(O)₂. The optionally substituted heteroaryl may be attached to its respective C2 or C4 nitrogen atom or linker L¹ or L² through any available carbon atom or heteroatom, but is typically attached via a carbon atom. The optional substituents may be the same or different, and may be attached to any available carbon atom or heteroatom. In one embodiment of these compounds, R⁵ is other than bromo, nitro, trifluoromethyl, cyano or —C(O)NHR, where R is hydrogen or (C1-C6) alkyl. In another embodiment of these compounds, when R² and R⁴ are each a substituted or unsubstituted pyrrole or indole, then the ring is attached to the remainder of the molecule via a ring carbon atom. In still another embodiment of compounds including an optionally substituted heteroaryl group, the heteroaryl is unsubstituted or substituted with from one to four of the same or different R⁸ groups, where R⁸ is as previously defined for structural formula (I). Specific examples of such optionally substituted heteroaryls include, but are not limited to, the following heteroaryl groups:

wherein:

p is an integer from one to three;

each - - - independently represents a single bond or a double bond;

R³⁵ is hydrogen or R⁸, where R⁸ is as previously defined for structural formula (I);

X is selected from the group consisting of CH, N and N—O ;

each Y is independently selected from the group consisting of O, S and NH;

each Y¹ is independently selected from the group consisting of O, S, SO, SO₂, SONR³⁶, NH and NR³⁷;

each Y² is independently selected from the group consisting of CH, CH₂, O, S, N, NH and NR³⁷;

R³⁶ is hydrogen or alkyl;

R³⁷ is selected from the group consisting of hydrogen and a progroup, preferably hydrogen or a progroup selected from the group consisting of aryl, arylalkyl, heteroaryl, R^(a), R^(b)—CR^(a)R^(b)—O—C(O)R⁸, —CR^(a)R^(b)—O—PO(OR⁸)₂, —CH₂—O—PO(OR⁸)₂, —CH₂—PO(OR⁸)₂, —C(O)—CR^(a)R^(b)—N(CH₃)₂, —CR^(a)R^(b)—O—C(O)—CR^(a)R^(b)—N(CH₃)₂, —C(O)R⁸, —C(O)CF₃ and —C(O)—NR⁸—C(O)R⁸;

A is selected from the group consisting of O, NH and NR³⁸;

R³⁸ is selected from the group consisting of alkyl and aryl;

R⁹, R¹⁰, R¹¹ and R¹² are each, independently of one another, selected from the group consisting of alkyl, alkoxy, halogen, haloalkoxy, aminoalkyl and hydroxyalkyl, or, alternatively, R⁹ and R¹⁰ and/or R¹¹ and R¹² are taken together form a ketal;

each Z is selected from the group consisting of hydroxyl, alkoxy, aryloxy, ester, carbamate and sulfonyl;

Q is selected from the group consisting of —OH, OR⁸, —NR^(c)R^(c), —NHR³⁹—C(O)R⁸, —NHR³⁹—C(O)OR⁸, —NR³⁹—CHR⁴⁰—R^(b), —NR³⁹—(CH₂)_(m)—R^(b) and —NR³⁹—C(O)—CHR⁴⁰—NR^(c)R^(c);

R³⁹ and R⁴⁰ are each, independently of one another, selected from the group consisting of hydrogen, alkyl, aryl, alkylaryl; arylalkyl and NHR⁸; and

R^(a), R^(b) and R^(c) are as previously defined for structural formula (I). Preferred R^(b) substitutents for Q are selected from —C(O)OR⁸, —O—C(O)R⁸, —O—P(O)(OR⁸)₂ and —P(O)(OR⁸)₂.

In one embodiment of the above-depicted heteroaryls, as well as other 5-15 membered heteroaryls according to this embodiment of the invention, each R⁸ is independently selected from the group consisting of R^(d), —NR^(c)R^(c), —(CH₂)_(m)—NR^(c)R^(c), —C(O)NR^(c)R^(c), —(CH₂)_(m)—C(O)NR^(c)R^(c), —C(O)OR^(d), —(CH₂)_(m)—C(O)OR^(d) and —(CH₂)_(m)—OR^(d), where m, R^(c) and R^(d) are as previously defined for structural formula (I).

In a specific embodiment, R^(d) and/or R^(c) is selected from the group consisting of R^(a) and (C3-C8) cycloalkyl optionally substituted with one or more of the same or different hydroxyl, amino or carboxyl groups.

In another embodiment of the above-depicted heteroaryls, each R³⁵ is a hydrogen atom, a (C1-C6) carbon chain, including methyl, ethyl, isopropyl, a cycloalkyl group, including cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, a

wherein x=1-8, —CH₂CONHMe, —CH₂CH₂NHMe, —CH₂CH₂CONHMe, —CH₂CH₂CH₂NHMe or —CH₂CH₂CH₂OCH₃.

In still another embodiment of the above-depicted heteroaryls, the aromatic ring connectivity is either at the 5 or 6 position. It should be understood that either R² or R⁴ can utilize the heteroaryl groups discussed throughout this specification.

In a fourteenth embodiment of the compounds of structural formulae (I) and (Ia), R² and R⁴ are each, independently of one another, an optionally substituted phenyl, aryl or heteroaryl, with the provisos that: (1) when L¹ is a direct bond and R⁶ and optionally R⁵ is hydrogen, then R² is other than 3,4,5-trimethoxyphenyl or 3,4,5-tri(C1-C6) alkoxyphenyl; (2) when L¹ and L² are each a direct bond, R⁶ is hydrogen and R⁵ is halo, then R² and R⁴ are not each simultaneously 3,4,5-trimethoxyphenyl or 3,4,5-tri(C1-C6) alkoxyphenyl; (3) when R⁴ is 3-methoxyphenyl or 3-(C1-C6) alkoxyphenyl and R² is a 3,4,5-trisubstituted phenyl, the substituents positioned at the 3 and 4 positions are not both simultaneously methoxy or (C1-C6) alkoxy; (4) when R² is a substituted phenyl and R⁶ is hydrogen, then R⁵ is other than cyano or —C(O)NHR, where R is hydrogen or (C1-C6) alkyl; and/or (5) when R² and R⁴ are each independently a substituted or unsubstituted pyrrole or indole, then the pyrrole or indole is attached to the remainder of the molecule via a ring carbon atom. Alternatively, R² is subject to the provisos described in connection with the first or second embodiment.

In this fourteenth embodiment of the invention, the R² and R⁴ substituents may be the same or different. Specific optionally substituted phenyl, aryl and/or heteroaryls include those illustrated above in connection with the twelfth and thirteenth embodiments.

In a fifteenth embodiment of the compounds of structural formulae (I) and (Ia), including the above-described first through fourteenth embodiments thereof, R⁶ is hydrogen and R⁵ is an electronegative group. As will be recognized by skilled artisans, electronegative groups are atoms or groups of atoms that have a relatively great tendency to attract electrons to themselves. Specific examples of electronegative groups according to this fourteenth embodiment include, but are not limited to, —CN, —NC, —NO₂, halo, bromo, chloro, fluoro, (C1-C3) haloalkyl, (C1-C3) perhaloalkyl, (C1-C3) fluoroalkyl, (C1-C3) perfluoroalkyl, —CF₃, (C1-C3) haloalkoxy, (C1-C3) perhaloalkoxy, (C1-C3) fluoroalkoxy, (C1-C3) perfluoroalkoxy, —OCF₃, —C(O)R^(a), —C(O)OR^(a), —C(O)CF₃ and —C(O)OCF₃. In a specific embodiment, the electronegative group is a halogen-containing electronegative group, such as —OCF₃, —CF₃, bromo, chloro or fluoro. In another specific embodiment, R⁵ is fluoro, subject to the proviso that the compound is not any compound according to the third embodiment.

In a sixteenth embodiment, the compounds of structural formulae (I) and (Ia) are compounds according to structural formula (Ib):

and salts, hydrates, solvates and N-oxides thereof, wherein R¹¹, R¹², R¹³ and R¹⁴ are each, independently of one another, selected from the group consisting of hydrogen, hydroxy, (C1-C6) alkoxy and —NR^(c)R^(c); and R⁵, R⁶ and R^(c) are as previously defined for structural formula (I), with the proviso that when R¹³, R⁵ and R⁶ are each hydrogen, then R¹¹ and R¹² are not simultaneously methoxy, (C1-C6) alkoxy or (C1-C6) haloalkoxy

In a seventeenth embodiment, the compounds of structural formulae (I) and (Ia) are compounds according to structural formula (Ic):

and salts, hydrates, solvates and N-oxides thereof, wherein:

R⁴ is selected from the group consisting of 5-10 membered heteroaryl and 3-hydroxyphenyl;

R⁵ is F or —CF₃; and

R⁸ is —O(CH₂)_(m)—R^(b), where m and R^(b) are as previously defined for structural formula (I). In a specific embodiment, R⁸ is —O—CH₂—C(O)NH—CH₃ and/or R⁴ is a heteroaryl according to the thirteenth embodiment.

In an eighteenth embodiment, the compounds of structural formulae (I) and (Ia) include any compound selected from TABLE 1 that inhibits an Fc receptor signal transduction cascade, a Syk kinase activity, a Syk-kinase dependent receptor signal transduction cascade or cell degranulation as measured in an in vitro assay, optionally subject to the proviso that the compound is not a compound excluded by the above-described third embodiment and/or other embodiments. In a specific embodiment, such compounds have an IC₅₀ of about 20 μM or less as measured in an in vitro degranulation assay, such as one of the degranulation assays described in the Examples section.

In a nineteenth embodiment, the compounds of structural formulae (I) and (Ia) include any compound selected from TABLE 1 that inhibits the FcγR1 or FcεR1 receptor cascade with an IC₅₀ of about 20 μM or less as measured in an in vitro assay, such as one of the in vitro assays provided in the Examples section, optionally subject to the proviso that the compound is not a compound excluded by the above-described third embodiment and/or other embodiments.

In a twentieth embodiment, the compounds of structural formulae (Ia) are those wherein R² is selected from the group consisting of

R⁴, R⁸, R^(a), R^(b), R^(c), R^(d) are as described above, R⁵ is a fluorine atom; R⁶ is a hydrogen atom and each R²¹ is independently a halogen atoms or an alkyl optionally substituted with one or more of the same or different halo groups, R²² and R²³ are each, independently of one another, a hydrogen atom, methyl or ethyl optionally substituted with one or more of the same or different halo groups, each m is independently an integer from 1 to 3, and each n is independently an integer from 0 to 3.

In a twenty first embodiment, the compounds of structural formulae (Ia) are those wherein R⁴ is

wherein R⁹ and R¹⁶ are as defined above and further include, each independently a hydrogen atom, and R² is a phenyl group, substituted with one or more of the same R⁸ groups, or

wherein R³⁵ is as defined above. In one particular aspect, when R² is a phenyl group, one or more of R⁸ is selected from a halogen and an alkoxy group. In one aspect, the phenyl group is di or tri substituted with one or more of the same R⁸ groups.

In a twenty second embodiment, the compounds of structural formulae (Ia) are those wherein R⁴ is

and R² is a phenyl group, substituted with one or more of the same R⁸ groups. In one particular aspect, one or more of R⁸ is selected from a halogen and an alkoxy group. In one aspect, the phenyl group is di or tri substituted with one or more of the same R⁸ groups.

In a twenty third embodiment, the compounds of structural formulae (Ia) are those wherein R⁴ is a phenyl group substituted with one or more of the same R⁸ groups, wherein R² is

wherein R³⁵ is as defined above. In particular embodiments, the R⁴ phenyl group is di or tri substituted with the same or different halogen atoms. In another embodiment, R⁴ is a monosubstituted phenyl group with a halogen atom. In one aspect, R³⁵ is a hydroxyalkyl group. In certain aspects, the hydroxyalkyl group can be further functionalized into an ester group, carbamate, etc.

In a twenty fourth embodiment, the compounds of structural formulae (Ia) are those wherein R⁴ is

wherein R³⁵ is as defined above and R² is a phenyl group substituted with one or more of the same R⁸ groups. In one particular aspect, R³⁵ is a hydrogen atom or an alkyl group. In another aspect, the R² phenyl group is di or tri substituted with the same or different R⁸ groups, and in particular, halogen atoms.

In a twenty fifth embodiment, the compounds of structural formulae (Ia) are those wherein R⁴ is

wherein R³⁵ is as defined above and R² is

wherein R⁹ and R¹⁰ are defined as above and further include, each independently a hydrogen atom. In one aspect, R³⁵ is a hydrogen atom or an alkyl group, e.g., methyl and R⁹ and R¹⁰ are alkyl groups, e.g., methyl groups.

In a twenty sixth embodiment, the compounds of structural formulae (Ia) are those wherein R⁴ is a disubstituted phenyl group, substituted with the same or different R⁸ groups and R² is

wherein R³⁵ is as defined above. In certain aspects, the phenyl group is substituted with a halogen atom and an alkyoxy group, e.g. a methoxy group. In certain embodiments, R³⁵ is a hydrogen atom, an alkyl group, e.g., a methyl group, or a hydroxyalkyl group. In certain aspects, the hydroxyalkyl group can be further functionalized into an ester group, carbamate, etc.

In a twenty seventh embodiment, the compounds of structural formulae (Ia) are those wherein R⁴ is

wherein R⁸ and R^(c) are as defined above and R² is a phenyl group that is substituted with one or more of the same R⁸ groups. In one particular aspect, R^(c) is a hydrogen atom or an alkyl group. In another aspect, the R² phenyl group is di or tri substituted with the same or different R⁸ groups, and in particular, halogen atoms or

In a twenty eighth embodiment, the compounds of structural formulae (Ia) are those wherein R⁴ is

wherein Y¹, Y² and each R³⁵ independently, are defined as above and R² is

wherein R³⁵ is as defined above. In one aspect of the twenty eighth embodiment with regard to R⁴, Y¹ is oxygen, Y² is NH and one or more of R³⁵ or the R⁴ moiety is an alkyl group, and in particular, a methyl group. In certain aspects of the twenty eighth embodiment, two R³⁵'s of the R⁴ moiety form a gem dialkyl moiety, in particular, a gem dimethyl moiety adjacent to the NH depicted as

In certain aspects of the twenty eighth embodiment, with regard to R², R³⁵ is a hydrogen atom or an alkyl group, and in particular, a methyl group.

In a twenty ninth embodiment, the compounds of structural formulae (Ia) are those wherein R⁴ is

wherein R⁹ and R¹⁰ are as defined above or a substituted phenyl group. In one aspect the phenyl group is di or tri substituted with one or more of the same R⁸ groups. In particular, the phenyl group can be di or tri substituted with one or more halogen atoms that can be the same or different. R² in the twenty ninth embodiment is

wherein R³⁵ is as defined above. In one aspect of the twenty ninth embodiment, R³⁵ of R² is not a methyl group. In another still another aspect of the twenty ninth embodiment, R² is

In a thirtieth embodiment, applicable to the first through twenty ninth embodiments, R⁵ is a halogen atom, such as fluorine, and R⁶ is a hydrogen atom.

Also specifically described are combinations of the above first through thirtieth embodiments.

Those of skill in the art will appreciate that the 2,4-pyrimidinediamine compounds described herein may include functional groups that can be masked with progroups to create prodrugs. Such prodrugs are usually, but need not be, pharmacologically inactive until converted into their active drug form. Indeed, many of the active 2,4-pyrimidinediamine compounds described in TABLE 1, infra, include promoieties that are hydrolyzable or otherwise cleavable under conditions of use. For example, ester groups commonly undergo acid-catalyzed hydrolysis to yield the parent carboxylic acid when exposed to the acidic conditions of the stomach, or base-catalyzed hydrolysis when exposed to the basic conditions of the intestine or blood. Thus, when administered to a subject orally, 2,4-pyrimidinediamines that include ester moieties may be considered prodrugs of their corresponding carboxylic acid, regardless of whether the ester form is pharmacologically active. Referring to TABLE 1, numerous ester-containing 2,4-pyrimidinediamines of the invention are active in their ester, “prodrug” form.

In the prodrugs of the invention, any available functional moiety may be masked with a progroup to yield a prodrug. Functional groups within the 2,4-pyrimidinediamine compounds that may be masked with progroups for inclusion in a promoiety include, but are not limited to, amines (primary and secondary), hydroxyls, sulfanyls (thiols), carboxyls, etc. Myriad progroups suitable for masking such functional groups to yield promoieties that are cleavable under the desired conditions of use are known in the art. All of these progroups, alone or in combinations, may be included in the prodrugs of the invention.

In one illustrative embodiment, the prodrugs of the invention are compounds according to structural formula (I) in which R^(c) and R^(d) may be, in addition to their previously-defined alternatives, a progroup.

Replacing the hydrogens attached to N2 and N4 in the 2,4-pyrimidinediamines of structural formula (I) with substituents adversely effects the activity of the compounds. However, as will be appreciated by skilled artisans, these nitrogens may be included in promoieties that, under conditions of use, cleave to yield 2,4-pyrimidinediamines according to structural formula (I). Thus, in another embodiment, the prodrugs of the invention are compounds according to structural formula (II):

including salts, hydrates, solvates and N-oxides thereof, wherein:

R², R⁴, R⁵, R⁶, L¹ and L² are as previously defined for structural formula (I); and

R^(2b) and R^(4b) are each, independently of one another, a progroup. Specific examples of progroups according to this embodiment of the invention include, but are not limited to, (C1-C6) alkyl, —C(O)CH₃, —C(O)NHR³⁶ and —S(O)₂R³⁶, where R³⁶ is (C1-C6) alkyl, (C5-C15) aryl and (C3-C8) cycloalkyl.

In the prodrugs of structural formula (II), the various substituents may be as described for the various first through twentieth embodiments previously described for the compounds of structural formulae (I) and (Ia), or combinations of such embodiments.

Those of skill in the art will appreciate that many of the compounds and prodrugs of the invention, as well as the various compound species specifically described and/or illustrated herein, may exhibit the phenomena of tautomerism, conformational isomerism, geometric isomerism and/or optical isomerism. For example, the compounds and prodrugs of the invention may include one or more chiral centers and/or double bonds and as a consequence may exist as stereoisomers, such as double-bond isomers (i.e., geometric isomers), enantiomers and diasteromers and mixtures thereof, such as racemic mixtures. As another example, the compounds and prodrugs of the invention may exist in several tautomeric forms, including the enol form, the keto form and mixtures thereof. As the various compound names, formulae and compound drawings within the specification and claims can represent only one of the possible tautomeric, conformational isomeric, optical isomeric or geometric isomeric forms, it should be understood that the invention encompasses any tautomeric, conformational isomeric, optical isomeric and/or geometric isomeric forms of the compounds or prodrugs having one or more of the utilities described herein, as well as mixtures of these various different isomeric forms. In cases of limited rotation around the 2,4-pryimidinediamine core structure, atrop isomers are also possible and are also specifically included in the compounds of the invention.

Moreover, skilled artisans will appreciate that when lists of alternative substituents include members which, owing to valency requirements or other reasons, cannot be used to substitute a particular group, the list is intended to be read in context to include those members of the list that are suitable for substituting the particular group. For example, skilled artisans will appreciate that while all of the listed alternatives for R^(b) can be used to substitute an alkyl group, certain of the alternatives, such as ═O, cannot be used to substitute a phenyl group. It is to be understood that only possible combinations of substituent-group pairs are intended.

The compounds and/or prodrugs of the invention may be identified by either their chemical structure or their chemical name. When the chemical structure and the chemical name conflict, the chemical structure is determinative of the identity of the specific compound.

Depending upon the nature of the various substituents, the 2,4-pyrimidinediamine compounds and prodrugs of the invention may be in the form of salts. Such salts include salts suitable for pharmaceutical uses (“pharmaceutically-acceptable salts”), salts suitable for veterinary uses, etc. Such salts may be derived from acids or bases, as is well-known in the art.

In one embodiment, the salt is a pharmaceutically acceptable salt. Generally, pharmaceutically acceptable salts are those salts that retain substantially one or more of the desired pharmacological activities of the parent compound and which are suitable for administration to humans. Pharmaceutically acceptable salts include acid addition salts formed with inorganic acids or organic acids. Inorganic acids suitable for forming pharmaceutically acceptable acid addition salts include, by way of example and not limitation, hydrohalide acids (e.g., hydrochloric acid, hydrobromic acid, hydriodic, etc.), sulfuric acid, nitric acid, phosphoric acid, and the like. Organic acids suitable for forming pharmaceutically acceptable acid addition salts include, by way of example and not limitation, acetic acid, trifluoroacetic acid, propionic acid, hexanoic acid, cyclopentanepropionic acid, glycolic acid, oxalic acid, pyruvic acid, lactic acid, malonic acid, succinic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, palmitic acid, benzoic acid, 3-(4-hydroxybenzoyl)benzoic acid, cinnamic acid, mandelic acid, alkylsulfonic acids (e.g., methanesulfonic acid, ethanesulfonic acid, 1,2-ethane-disulfonic acid, 2-hydroxyethanesulfonic acid, etc.), arylsulfonic acids (e.g., benzenesulfonic acid, 4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid, 4-toluenesulfonic acid, cycloalkylsulfonic acids (e.g., camphorsulfonic acid), 4-methylbicyclo[2.2.2]-oct-2-ene-1-carboxylic acid, glucoheptonic acid, 3-phenylpropionic acid, trimethylacetic acid, tertiary butylacetic acid, lauryl sulfuric acid, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylic acid, stearic acid, muconic acid, and the like.

Pharmaceutically acceptable salts also include salts formed when an acidic proton present in the parent compound is either replaced by a metal ion (e.g., an alkali metal ion, an alkaline earth metal ion or an aluminum ion), an ammonium ion or coordinates with an organic base (e.g., ethanolamine, diethanolamine, triethanolamine, N-methylglucamine, morpholine, piperidine, dimethylamine, diethylamine, etc.).

The 2,4-pyrimidinediamine compounds and of the invention, as well as the salts thereof, may also be in the form of hydrates, solvates and N-oxides, as are well-known in the art.

6.3 Methods of Synthesis

The compounds and prodrugs of the invention may be synthesized via a variety of different synthetic routes using commercially available starting materials and/or starting materials prepared by conventional synthetic methods. Suitable exemplary methods that may be routinely adapted to synthesize the 2,4-pyrimidinediamine compounds and prodrugs of the invention are found in U.S. Pat. No. 5,958,935, the disclosure of which is incorporated herein by reference. Specific examples describing the synthesis of numerous compounds and prodrugs of the invention, as well as intermediates therefor, are provided in the Examples section. All of the compounds of structural formulae (I), (Ia) and (II) may be prepared by routine adaptation of these methods.

A variety of exemplary synthetic routes that can be used to synthesize the 2,4-pyrimidinediamine compounds of the invention are described in Schemes (I)-(XI), below. In Schemes (I)-(XI), like-numbered compounds have similar structures. These methods may be routinely adapted to synthesize the prodrugs according to structural formula (II). In one exemplary embodiment, the compounds can be synthesized from substituted or unsubstituted uracils or thiouracils as illustrated in Scheme (I), below:

In Scheme (I), R², R⁴, R⁵, R⁶, L¹ and L² are as previously defined for structural formula (I), X is a halogen (e.g., F, Cl, Br or I) and Y and Y′ are each, independently of one another, selected from the group consisting of O and S. Referring to Scheme (I), uracil or thiouracil 2 is dihalogenated at the 2- and 4-positions using standard halogenating agent POX₃ (or other standard halogenating agent) under standard conditions to yield 2,4-bishalo pyrimidine 4. Depending upon the R⁵ substituent, in pyrimidine 4, the halide at the C4 position is more reactive towards nucleophiles than the halide at the C2 position. This differential reactivity can be exploited to synthesize 2,4-pyrimidinediamines according structural formula (I) by first reacting 2,4-bishalopyrimidine 4 with one equivalent of amine 10, yielding 4N-substituted-2-halo-4-pyrimidineamine 8, followed by amine 6 to yield a 2,4-pyrimidinediamine according structural formula (I). 2N,4N-bis(substituted)-2,4-pyrimidinediamines 12 and 14 can be obtained by reacting 2,4-bishalopyrimidine 4 with excess 6 or 10, respectively.

In most situations, the C4 halide is more reactive towards nucleophiles, as illustrated in the Scheme. However, as will be recognized by skilled artisans, the identity of the R⁵ substituent may alter this reactivity. For example, when R⁵ is trifluoromethyl, a 50:50 mixture of 4N-substituted-4-pyrimidineamine 8 and the corresponding 2N-substituted-2-pyrimidineamine is obtained. Regardless of the identity of the R⁵ substituent, the regioselectivity of the reaction can be controlled by adjusting the solvent and other synthetic conditions (such as temperature), as is well-known in the art.

The reactions depicted in Scheme (I) may proceed more quickly when the reaction mixtures are heated via microwave. When heating in this fashion, the following conditions may be used: heat to 175° C. in ethanol for 5-20 min. in a Smith Reactor (Personal Chemistry) in a sealed tube (at 20 bar pressure).

The uracil or thiouracil 2 starting materials may be purchased from commercial sources or prepared using standard techniques of organic chemistry. Commercially available uracils and thiouracils that can be used as starting materials in Scheme (I) include, by way of example and not limitation, uracil (Aldrich #13, 078-8; CAS Registry 66-22-8); 2-thio-uracil (Aldrich #11, 558-4; CAS Registry 141-90-2); 2,4-dithiouracil (Aldrich #15, 846-1; CAS Registry 2001-93-6); 5-acetouracil (Chem. Sources Int'l 2000; CAS Registry 6214-65-9); 5-azidouracil; 5-aminouracil (Aldrich #85, 528-6; CAS Registry 932-52-5); 5-bromouracil (Aldrich #85, 247-3; CAS Registry 51-20-7); 5-(trans-2-bromovinyl)-uracil (Aldrich #45, 744-2; CAS Registry 69304-49-0); 5-(trans-2-chlorovinyl)-uracil (CAS Registry 81751-48-2); 5-(trans-2-carboxyvinyl)-uracil; uracil-5-carboxylic acid (2,4-dihydroxypyrimidine-5-carboxylic acid hydrate; Aldrich #27, 770-3; CAS Registry 23945-44-0); 5-chlorouracil (Aldrich #22, 458-8; CAS Registry 1820-81-1); 5-cyanouracil (Chem. Sources Int'12000; CAS Registry 4425-56-3); 5-ethyluracil (Aldrich #23, 044-8; CAS Registry 4212-49-1); 5-ethenyluracil (CAS Registry 37107-81-6); 5-fluorouracil (Aldrich #85, 847-1; CAS Registry 51-21-8); 5-iodouracil (Aldrich #85, 785-8; CAS Registry 696-07-1); 5-methyluracil (thymine; Aldrich #13, 199-7; CAS Registry 65-71-4); 5-nitrouracil (Aldrich #85, 276-7; CAS Registry 611-08-5); uracil-5-sulfamic acid (Chem. Sources Int'12000; CAS Registry 5435-16-5); 5-(trifluoromethyl)-uracil (Aldrich #22, 327-1; CAS Registry 54-20-6); 5-(2,2,2-trifluoroethyl)-uracil (CAS Registry 155143-31-6); 5-(pentafluoroethyl)-uracil (CAS Registry 60007-38-3); 6-aminouracil (Aldrich #A5060-6; CAS Registry 873-83-6) uracil-6-carboxylic acid (orotic acid; Aldrich #0-840-2; CAS Registry 50887-69-9); 6-methyluracil (Aldrich #D11, 520-7; CAS Registry 626-48-2); uracil-5-amino-6-carboxylic acid (5-aminoorotic acid; Aldrich #19, 121-3; CAS Registry #7164-43-4); 6-amino-5-nitrosouracil (6-amino-2,4-dihydroxy-5-nitrosopyrimidine; Aldrich #27, 689-8; CAS Registry 5442-24-0); uracil-5-fluoro-6-carboxylic acid (5-fluoroorotic acid; Aldrich #42, 513-3; CAS Registry 00000-00-0); and uracil-5-nitro-6-carboxylic acid (5-nitroorotic acid; Aldrich #18, 528-0; CAS Registry 600779-49-9). Additional 5-, 6- and 5,6-substituted uracils and/or thiouracils are available from General Intermediates of Canada, Inc., Edmonton, Alberta, CA. (www.generalintermediates.com) and/or Interchim, France (www.interchim.com), or may be prepared using standard techniques. Myriad textbook references teaching suitable synthetic methods are provided infra.

Amines 6 and 10 may be purchased from commercial sources or, alternatively, may be synthesized utilizing standard techniques. For example, suitable amines may be synthesized from nitro precursors using standard chemistry. Specific exemplary reactions are provided in the Examples section. See also Vogel, 1989, Practical Organic Chemistry, Addison Wesley Longman, Ltd. and John Wiley & Sons, Inc.

Skilled artisans will recognize that in some instances, amines 6 and 10 and/or substituents R⁵ and/or R⁶ on uracil or thiouracil 2 may include functional groups that require protection during synthesis. The exact identity of any protecting group(s) used will depend upon the identity of the functional group being protected, and will be apparent to these of skill in the art. Guidance for selecting appropriate protecting groups, as well as synthetic strategies for their attachment and removal, may be found, for example, in Greene & Wuts, Protective Groups in Organic Synthesis, 3d Edition, John Wiley & Sons, Inc., New York (1999) and the references cited therein (hereinafter “Greene & Wuts”).

A specific embodiment of Scheme (I) utilizing 5-fluorouracil (Aldrich #32, 937-1) as a starting material is illustrated in Scheme (Ia), below:

In Scheme (Ia), R², R⁴, L¹ and L² are as previously defined for Scheme (I). According to Scheme (Ia), 5-fluorouracil 3 is halogenated with POCl₃ to yield 2,4-dichloro-5-fluoropyrimidine 5, which is then reacted with excess amine 6 or 10 to yield N2,N4-bis substituted 5-fluoro-2,4-pyrimidinediamine 11 or 13, respectively. Alternatively, asymmetric 2N,4N-disubstituted-5-fluoro-2,4-pyrimidinediamine 9 may be obtained by reacting 2,4-dichloro-5-fluoropyrimidine 5 with one equivalent of amine 10 (to yield 2-chloro-N4-substituted-5-fluoro-4-pyrimidineamine 7) followed by one or more equivalents of amine 6.

In another exemplary embodiment, the 2,4-pyrimidinediamine compounds of the invention may be synthesized from substituted or unsubstituted cytosines as illustrated in Schemes (IIa) and (IIb), below:

In Schemes (IIa) and (IIb), R², R⁴, R⁵, R⁶, L¹, L² and X are as previously defined for Scheme (I) and PG represents a protecting group. Referring to Scheme (IIa), the C4 exocyclic amine of cytosine 20 is first protected with a suitable protecting group PG to yield N4-protected cytosine 22. For specific guidance regarding protecting groups useful in this context, see Vorbrüggen and Ruh-Pohlenz, 2001, Handbook of Nucleoside Synthesis, John Wiley & Sons, NY, pp. 1-631 (“Vorbrüggen”). Protected cytosine 22 is halogenated at the C2 position using a standard halogenation reagent under standard conditions to yield 2-chloro-4N-protected-4-pyrimidineamine 24. Reaction with amine 6 followed by deprotection of the C4 exocyclic amine and reaction with amine 10 yields a 2,4-pyrimidinediamine according to structural formula (I).

Alternatively, referring to Scheme (IIb), cytosine 20 may be reacted with amine 10 or protected amine 21 to yield N4-substituted cytosine 23 or 27, respectively. These substituted cytosines may then be halogenated as previously described, deprotected (in the case of N4-substituted cytosine 27) and reacted with amine 6 to yield a 2,4-pyrimidinediamine according to structural formula (I).

Commercially-available cytosines that may be used as starting materials in Schemes (IIa) and (IIb) include, but are not limited to, cytosine (Aldrich #14, 201-8; CAS Registry 71-30-7); N⁴-acetylcytosine (Aldrich #37, 791-0; CAS Registry 14631-20-0); 5-fluorocytosine (Aldrich #27, 159-4; CAS Registry 2022-85-7); and 5-(trifluoromethyl)-cytosine. Other suitable cytosines useful as starting materials in Schemes (IIa) are available from General Intermediates of Canada, Inc., Edmonton, Alberta, Calif. (www.generalintermediates.com) and/or Interchim, France (www.interchim.com), or may be prepared using standard techniques. Myriad textbook references teaching suitable synthetic methods are provided infra.

In still another exemplary embodiment, the 2,4-pyrimidinediamine compounds of the invention may be synthesized from substituted or unsubstituted 2-amino-4-pyrimidinols as illustrated in Scheme (III), below:

In Scheme (III), R², R⁴, R⁵, R⁶, L¹, L² and X are as previously defined for Scheme (I) and Z is a leaving group as discussed in more detail in connection with Scheme IV, infra. Referring to Scheme (III), 2-amino-4-pyrimidinol 30 is reacted with amine 6 (or optionally protected amine 21) to yield N2-substituted-4-pyrimidinol 32, which is then halogenated as previously described to yield N2-substituted-4-halo-2-pyrimidineamine 34. Optional deprotection (for example if protected amine 21 was used in the first step) followed by reaction with amine 10 affords a 2,4-pyrimidinediamine according to structural formula (I). Alternatively, pyrimidinol 30 can be reacted with acylating agent 31.

Suitable commercially-available 2-amino-4-pyrimidinols 30 that can be used as starting materials in Scheme (III) include, but are not limited to, 2-amino-6-chloro-4-pyrimidinol hydrate (Aldrich #A4702-8; CAS Registry 00000-00-0) and 2-amino-6-hydroxy-4-pyrimidinol (Aldrich #A5040-1; CAS Registry 56-09-7). Other 2-amino-4-pyrimidinols 30 useful as starting materials in Scheme (III) are available from General Intermediates of Canada, Inc., Edmonton, Alberta, CA (www.generalintermediates.com) and/or Interchim, France (www.interchim.com), or may be prepared using standard techniques. Myriad textbook references teaching suitable synthetic methods are provided infra.

Alternatively, the 2,4-pyrimidinediamine compounds of the invention may be prepared from substituted or unsubstituted 4-amino-2-pyrimidinols as illustrated in Scheme (IV), below:

In Scheme (IV), R², R⁴, R⁵, R⁶, L¹ and L² are as previously defined for Scheme (I) and Z represents a leaving group. Referring to Scheme (IV), the C2-hydroxyl of 4-amino-2-pyrimidinol 40 is more reactive towards nucleophiles than the C4-amino such that reaction with amine 6 yields N2-substituted-2,4-pyrimidinediamine 42. Subsequent reaction with compound 44, which includes a good leaving group Z, or amine 10 yields a 2,4-pyrimidinediamine according to structural formula (I). Compound 44 may include virtually any leaving group that can be displaced by the C4-amino of N2-substituted-2,4-pyrimidinediamine 42. Suitable leaving groups Z include, but are not limited to, halogens, methanesulfonyloxy (mesyloxy; “OMs”), trifluoromethanesulfonyloxy (“OTf”) and p-toluenesulfonyloxy (tosyloxy; “OTs”), benzene sulfonyloxy (“besylate”) and metanitro benzene sulfonyloxy (“nosylate”). Other suitable leaving groups will be apparent to those of skill in the art.

Substituted 4-amino-2-pyrimidinol starting materials may be obtained commercially or synthesized using standard techniques. Myriad textbook references teaching suitable synthetic methods are provided infra.

In still another exemplary embodiment, the 2,4-pyrimidinediamine compounds of the invention can be prepared from 2-chloro-4-aminopyrimidines or 2-amino-4-chloropyrimidines as illustrated in Scheme (V), below:

In Scheme (V), R², R⁴, R⁵, R⁶, L¹, L² and X are as defined for Scheme (I) and Z is as defined for Scheme (IV). Referring to Scheme (V), 2-amino-4-chloropyrimidine 50 is reacted with amino 10 to yield 4N-substituted-2-pyrimidineamine 52 which, following reaction with compound 31 or amine 6, yields a 2,4-pyrimidinediamine according to structural formula (I). Alternatively, 2-chloro-4-amino-pyrimidine 54 may be reacted with compound 44 followed by amine 6 to yield a compound according to structural formula (I).

A variety of pyrimidines 50 and 54 suitable for use as starting materials in Scheme (V) are commercially available, including by way of example and not limitation, 2-amino-4,6-dichloropyrimidine (Aldrich #A4860-1; CAS Registry 56-05-3); 2-amino-4-chloro-6-methoxy-pyrimidine (Aldrich #51, 864-6; CAS Registry 5734-64-5); 2-amino-4-chloro-6-methylpyrimidine (Aldrich #12, 288-2; CAS Registry 5600-21-5); and 2-amino-4-chloro-6-methylthiopyrimidine (Aldrich #A4600-5; CAS Registry 1005-38-5). Additional pyrimidine starting materials are available from General Intermediates of Canada, Inc., Edmonton, Alberta, CA (www.generalintermediates.com) and/or Interchim, France (www.interchim.com), or may be prepared using standard techniques. Myriad textbook references teaching suitable synthetic methods are provided infra.

Alternatively, 4-chloro-2-pyrimidineamines 50 may be prepared as illustrated in Scheme (Va):

In Scheme (Va), R⁵ and R⁶ are as previously defined for structural formula (I). In Scheme (Va), dicarbonyl 53 is reacted with guanidine to yield 2-pyrimidineamine 51. Reaction with peracids like m-chloroperbenzoic acid, trifluoroperacetic acid or urea hydrogen peroxide complex yields N-oxide 55, which is then halogenated to give 4-chloro-2-pyrimidineamine 50. The corresponding 4-halo-2-pyrimidineamines may be obtained by using suitable halogenation reagents.

In yet another exemplary embodiment, the 2,4-pyrimidinediamine compounds of the invention can be prepared from substituted or unsubstituted uridines as illustrated in Scheme (VI), below:

In Scheme (VI), R², R⁴, R⁵, R⁶, L¹, L² and X are as previously defined for Scheme (I) and the superscript PG represents a protecting group, as discussed in connection with Scheme (IIb). According to Scheme (VI), uridine 60 has a C4 reactive center such that reaction with amine 10 or protected amine 21 yields N4-substituted cytidine 62 or 64, respectively. Acid-catalyzed deprotection of N4-substituted 62 or 64 (when “PG” represents an acid-labile protecting group) yields N4-substituted cytosine 28, which may be subsequently halogenated at the C2-position and reacted with amine 6 to yield a 2,4-pyrimidinediamine according to structural formula (I).

Cytidines may also be used as starting materials in an analogous manner, as illustrated in Scheme (VII), below:

In Scheme (VII), R², R⁴, R⁵, R⁶, L¹, L² and X are as previously defined in Scheme (I) and the superscript PG represents a protecting group as discussed above. Referring to Scheme (VII), like uridine 60, cytidine 70 has a C4 reactive center such that reaction with amine 10 or protected amine 21 yields N4-substituted cytidine 62 or 64, respectively. These cytidines 62 and 64 are then treated as previously described for Scheme (VI) to yield a 2,4-pyrimidinediamine according to structural formula (I).

Although Schemes (VI) and (VII) are exemplified with ribosylnucleosides, skilled artisans will appreciate that the corresponding 2′-deoxyribo and 2′,3′-dideoxyribo nucleosides, as well as nucleosides including sugars or sugar analogs other than ribose, would also work.

Numerous uridines and cytidines useful as starting materials in Schemes (VI) and (VII) are known in the art, and include, by way of example and not limitation, 5-trifluoromethyl-2′-deoxycytidine (Chem. Sources #ABCR F07669; CAS Registry 66, 384-66-5); 5-bromouridine (Chem. Sources Int'l 2000; CAS Registry 957-75-5); 5-iodo-2′-deoxyuridine (Aldrich #1-775-6; CAS Registry 54-42-2); 5-fluorouridine (Aldrich #32, 937-1; CAS Registry 316-46-1); 5-iodouridine (Aldrich #85, 259-7; CAS Registry 1024-99-3); 5-(trifluoromethyl)uridine (Chem. Sources Int'12000; CAS Registry 70-00-8); 5-trifluoromethyl-2′-deoxyuridine (Chem. Sources Int'12000; CAS Registry 70-00-8). Additional uridines and cytidines that can be used as starting materials in Schemes (VI) and (VII) are available from General Intermediates of Canada, Inc., Edmonton, Alberta, CA (www.generalintermediates.com) and/or Interchim, France (www.interchim.com), or may be prepared using standard techniques. Myriad textbook references teaching suitable synthetic methods are provided infra.

The 2,4-pyrimidinediamine compounds of the invention can also be synthesized from substituted pyrimidines, such as chloro-substituted pyrimidines, as illustrated in Schemes (VIII) and (IX), below:

In Schemes (VIII) and (IX), R², R⁴, L¹, L² and R^(a) are as previously defined for structural formula (I) and “Ar” represents an aryl group. Referring to Scheme (VIII), reaction of 2,4,6-trichloropyrimidine 80 (Aldrich #T5, 620-0; CAS#3764-01-0) with amine 6 yields a mixture of three compounds: substituted pyrimidine mono-, di- and triamines 81, 82 and 83, which can be separated and isolated using HPLC or other conventional techniques. Mono- and diamines 81 and 82 may be further reacted with amines 6 and/or 10 to yield N2,N4,N6-trisubstituted-2,4,6-pyrimidinetriamines 84 and 85, respectively. N2,N4-bis-substituted-2,4-pyrimidinediamines can be prepared in a manner analogous to Scheme (VIII) by employing 2,4-dichloro-5-methylpyrimidine or 2,4-dichloro-pyrimidine as starting materials. In this instance, the mono-substituted pyrimidineamine corresponding to compound 81 is not obtained. Instead, the reaction proceeds to yield the N2,N4-bis-substituted-2,4-pyrimidinediamine directly.

Referring to Scheme (IX), 2,4,5,6-tetrachloropyrimidine 90 (Aldrich #24,671-9; CAS#1780-40-1) is reacted with excess amine 6 to yield a mixture of three compounds: 91, 92, and 93, which can be separated and isolated using HPLC or other conventional techniques. As illustrated, N2,N4-bis-substituted-5,6,-dichloro-2,4-pyrimidinediamine 92 may be further reacted at the C6 halide with, for example a nucleophilic agent 94 to yield compound 95. Alternatively, compound 92 can be converted into N2,N4-bis-substituted-5-chloro-6-aryl-2,4-pyrimidinediamine 97 via a Suzuki reaction. 2,4-Pyrimidinediamine 95 may be converted to 2,4-pyrimidinediamine 99 by reaction with Bn₃SnH.

As will be recognized by skilled artisans, 2,4-pyrimidinediamines according to the invention, synthesized via the exemplary methods described above or by other well-known means, may also be utilized as starting materials and/or intermediates to synthesize additional 2,4-pyrimidinediamine compounds of the invention. A specific example is illustrated in Scheme (X), below:

In Scheme (X), R⁴, R⁵, R⁶, L² and R^(a) are as previously defined for structural formula (I). Each R^(a) is independently an R^(a), and may be the same or different from the illustrated R^(a). Referring to Scheme (X), carboxylic acid or ester 100 may be converted to amide 104 by reaction with amine 102. In amine 102, R^(a) may be the same or different than R^(a) of acid or ester 100. Similarly, carbonate ester 106 may be converted to carbamate 108.

A second specific example is illustrated in Scheme (XI), below:

In Scheme (XI), R⁴, R⁵, R⁶, L² and R^(c) are as previously defined for structural formula (I). Referring to Scheme (XI), amide 110 or 116 may be converted to amine 114 or 118, respectively, by borane reduction with borane methylsulfide complex 112. Other suitable reactions for synthesizing 2,4-pyrimidinediamine compounds from 2,4-pyrimidinediamine starting materials will be apparent to those of skill in the art.

Although many of the synthetic schemes discussed above do not illustrate the use of protecting groups, skilled artisans will recognize that in some instances substituents R², R⁴, R⁵, R⁶, L¹ and/or L² may include functional groups requiring protection. The exact identity of the protecting group used will depend upon, among other things, the identity of the functional group being protected and the reaction conditions used in the particular synthetic scheme, and will be apparent to those of skill in the art. Guidance for selecting protecting groups and chemistries for their attachment and removal suitable for a particular application can be found, for example, in Greene & Wuts, supra.

Prodrugs according to structural formula (II) may be prepared by routine modification of the above-described methods. Alternatively, such prodrugs may be prepared by reacting a suitably protected 2,4-pyrimidinediamine of structural formula (I) with a suitable progroup. Conditions for carrying out such reactions and for deprotecting the product to yield a prodrug of formula (II) are well-known.

Myriad references teaching methods useful for synthesizing pyrimidines generally, as well as starting materials described in Schemes (I)-(IX), are known in the art. For specific guidance, the reader is referred to Brown, D. J., “The Pyrimidines”, in The Chemistry of Heterocyclic Compounds, Volume 16 (Weissberger, A., Ed.), 1962, Interscience Publishers, (A Division of John Wiley & Sons), New York (“Brown I”); Brown, D. J., “The Pyrimidines”, in The Chemistry of Heterocyclic Compounds, Volume 16, Supplement I (Weissberger, A. and Taylor, E. C., Ed.), 1970, Wiley-Interscience, (A Division of John Wiley & Sons), New York (Brown II″); Brown, D. J., “The Pyrimidines”, in The Chemistry of Heterocyclic Compounds, Volume 16, Supplement II (Weissberger, A. and Taylor, E. C., Ed.), 1985, An Interscience Publication (John Wiley & Sons), New York (“Brown III”); Brown, D. J., “The Pyrimidines” in The Chemistry of Heterocyclic Compounds, Volume 52 (Weissberger, A. and Taylor, E. C., Ed.), 1994, John Wiley & Sons, Inc., New York, pp. 1-1509 (Brown IV″); Kenner, G. W. and Todd, A., in Heterocyclic Compounds, Volume 6, (Elderfield, R. C., Ed.), 1957, John Wiley, New York, Chapter 7 (pyrimidines); Paquette, L. A., Principles of Modern Heterocyclic Chemistry, 1968, W. A. Benjamin, Inc., New York, pp. 1-401 (uracil synthesis pp. 313, 315; pyrimidine synthesis pp. 313-316; amino pyrimidine synthesis pp. 315); Joule, J. A., Mills, K. and Smith, G. F., Heterocyclic Chemistry, 3^(rd) Edition, 1995, Chapman and Hall, London, UK, pp. 1-516; Vorbrüggen, H. and Ruh-Pohlenz, C., Handbook of Nucleoside Synthesis, John Wiley & Sons, New York, 2001, pp. 1-631 (protection of pyrimidines by acylation pp. 90-91; silylation of pyrimidines pp. 91-93); Joule, J. A., Mills, K. and Smith, G. F., Heterocyclic Chemistry, 4^(th) Edition, 2000, Blackwell Science, Ltd, Oxford, UK, pp. 1-589; and Comprehensive Organic Synthesis, Volumes 1-9 (Trost, B. M. and Fleming, I., Ed.), 1991, Pergamon Press, Oxford, UK.

6.4 Inhibition of Fc Receptor Signal Cascades

Active 2,4-pyrimidinediamine compounds of the invention inhibit Fc receptor signalling cascades that lead to, among other things, degranulation of cells. As a specific example, the compounds inhibit the FcεRI and/or FcγRI signal cascades that lead to degranulation of immune cells such as neutrophil, eosinophil, mast and/or basophil cells. Both mast and basophil cells play a central role in allergen-induced disorders, including, for example, allergic rhinitis and asthma. Referring to FIG. 1, upon exposure allergens, which may be, among other things, pollen or parasites, allergen-specific IgE antibodies are synthesized by B-cells activated by IL-4 (or IL-13) and other messengers to switch to IgE class specific antibody synthesis. These allergen-specific IgEs bind to the high affinity FcεRI. Upon binding of antigen, the FcεR1-bound IgEs are cross-linked and the IgE receptor signal transduction pathway is activated, which leads to degranulation of the cells and consequent release and/or synthesis of a host of chemical mediators, including histamine, proteases (e.g., tryptase and chymase), lipid mediators such as leukotrienes (e.g., LTC4), platelet-activating factor (PAF) and prostaglandins (e.g., PGD2) and a series of cytokines, including TNF-α, IL-4, IL-13, IL-5, IL-6, IL-8, GMCSF, VEGF and TGF-β. The release and/or synthesis of these mediators from mast and/or basophil cells accounts for the early and late stage responses induced by allergens, and is directly linked to downstream events that lead to a sustained inflammatory state.

The molecular events in the FcεRI signal transduction pathway that lead to release of preformed mediators via degranulation and release and/or synthesis of other chemical mediators are well-known and are illustrated in FIG. 2. Referring to FIG. 2, the FcεRI is a heterotetrameric receptor composed of an IgE-binding alpha-subunit, a beta subunit, and two gamma subunits (gamma homodimer). Cross-linking of FcεRI-bound IgE by multivalent binding agents (including, for example IgE-specific allergens or anti-IgE antibodies or fragments) induces the rapid association and activation of the Src-related kinase Lyn. Lyn phosphorylates immunoreceptor tyrosine-based activation motifs (ITAMS) on the intracellular beta and gamma subunits, which leads to the recruitment of additional Lyn to the beta subunit and Syk kinase to the gamma homodimer. These receptor-associated kinases, which are activated by intra- and intermolecular phosphorylation, phosphorylate other components of the pathway, such as the Btk kinase, LAT, and phospholipase C-gamma PLC-gamma) Activated PLC-gamma initiates pathways that lead to protein kinase C activation and Ca²⁺ mobilization, both of which are required for degranulation. FcεR1 cross-linking also activates the three major classes of mitogen activated protein (MAP) kinases, i.e. ERK1/2, JNK1/2, and p38. Activation of these pathways is important in the transcriptional regulation of proinflammatory mediators, such as TNF-α and IL-6, as well as the lipid mediator leukotriene CA (LTC4).

Although not illustrated, the FcγRI signaling cascade is believed to share some common elements with the FceRI signaling cascade. Importantly, like FcεRI, the FcγRI includes a gamma homodimer that is phosphorylated and recruits Syk, and like FcεRI, activation of the FcγRI signaling cascade leads to, among other things, degranulation. Other Fc receptors that share the gamma homodimer, and which can be regulated by the active 2,4-pyrimidinediamine compounds include, but are not limited to, FcαRI and FcγRIII.

The ability of the 2,4-pyrimidinediamine compounds of the invention to inhibit Fc receptor signaling cascades may be simply determined or confirmed in in vitro assays. Suitable assays for confirming inhition of FcεRI-mediated degranulation are provided in the Examples section. In one typical assay, cells capable of undergoing FcεRI-mediated degranulation, such as mast or basophil cells, are first grown in the presence of IL-4, Stem Cell Factor (SCF), IL-6 and IgE to increase expression of the FcεRI, exposed to a 2,4-pyrimidinediamine test compound of the invention and stimulated with anti-IgE antibodies (or, alternatively, an IgE-specific allergen). Following incubation, the amount of a chemical mediator or other chemical agent released and/or synthesized as a consequence of activating the FcεRI signaling cascade may be quantified using standard techniques and compared to the amount of the mediator or agent released from control cells (i.e., cells that are stimulated but that are not exposed to test compound). The concentration of test compound that yields a 50% reduction in the quantity of the mediator or agent measured as compared to control cells is the IC₅₀ of the test compound. The origin of the mast or basophil cells used in the assay will depend, in part, on the desired use for the compounds and will be apparent to those of skill in the art. For example, if the compounds will be used to treat or prevent a particular disease in humans, a convenient source of mast or basophil cells is a human or other animal which constitutes an accepted or known clinical model for the particular disease. Thus, depending upon the particular application, the mast or basophil cells may be derived from a wide variety of animal sources, ranging from, for example, lower mammals such as mice and rats, to dogs, sheep and other mammals commonly employed in clinical testing, to higher mammals such as monkeys, chimpanzees and apes, to humans. Specific examples of cells suitable for carrying out the in vitro assays include, but are not limited to, rodent or human basophil cells, rat basophil leukemia cell lines, primary mouse mast cells (such as bone marrow-derived mouse mast cells “BMMC”) and primary human mast cells isolated from cord blood (“CHMC”) or other tissues such as lung. Methods for isolating and culturing these cell types are well-known or are provided in the Examples section (see, e.g., Demo et al., 1999, Cytometry 36(4):340-348 and copending application Ser. No. 10/053,355, filed Nov. 8, 2001, the disclosures of which are incorporated herein by reference). Of course, other types of immune cells that degranulate upon activation of the FcεRI signaling cascade may also be used, including, for example, eosinophils.

As will be recognized by skilled artisans, the mediator or agent quantified is not critical. The only requirement is that it be a mediator or agent released and/or synthesized as a consequence of initiating or activating the Fc receptor signaling cascade. For example, referring to FIG. 1, activation of the FcεRI signaling cascade in mast and/or basophil cells leads to numerous downstream events. For example, activation of the FcεRI signal cascade leads to the immediate release (i.e., within 1-3 min. following receptor activation) of a variety of preformed chemical mediators and agents via degranulation. Thus, in one embodiment, the mediator or agent quantified may be specific to granules (i.e., present in granules but not in the cell cytoplasm generally). Examples of granule-specific mediators or agents that can be quantified to determine and/or confirm the activity of a 2,4-pyrimidinediamine compound of the invention include, but are not limited to, granule-specific enzymes such as hexosaminidase and tryptase and granule-specific components such as histamine and serotonin. Assays for quantifying such factors are well-known, and in many instances are commercially available. For example, tryptase and/or hexosaminidase release may be quantified by incubating the cells with cleavable substrates that fluoresce upon cleavage and quantifying the amount of fluorescence produced using conventional techniques. Such cleavable fluorogenic substrates are commercially available. For example, the fluorogenic substrates Z-Gly-Pro-Arg-AMC (Z=benzyloxycarbonyl; AMC=7-amino-4-methylcoumarin; BIOMOL Research Laboratories, Inc., Plymouth Meeting, Pa. 19462, Catalog No. P-142) and Z-Ala-Lys-Arg-AMC (Enzyme Systems Products, a division of ICN Biomedicals, Inc., Livermore, CA 94550, Catalog No. AMC-246) can be used to quantify the amount of tryptase released. The fluorogenic substrate 4-methylumbelliferyl-N-acetyl-13-D-glucosaminide (Sigma, St. Louis, Mo., Catalog #69585) can be used to quantify the amount of hexosaminidase released. Histamine release may be quantified using a commercially available enzyme-linked immunosorbent assay (ELISA) such as Immunotech histamine ELISA assay #IM2015 (Beckman-Coulter, Inc.). Specific methods of quantifying the release of tryptase, hexosaminidase and histamine are provided in the Examples section. Any of these assays may be used to determine or confirm the activity of the 2,4-pyrimidinediamine compounds of the invention.

Referring again to FIG. 1, degranulation is only one of several responses initiated by the FcεRI signaling cascade. In addition, activation of this signaling pathway leads to the de novo synthesis and release of cytokines and chemokines such as IL-4, IL-5, IL-6, TNF-α, IL-13 and MIP1-α), and release of lipid mediators such as leukotrienes (e.g., LTC4), platelet activating factor (PAF) and prostaglandins. Accordingly, the 2,4-pyrimidinediamine compounds of the invention may also be assessed for activity by quantifying the amount of one or more of these mediators released and/or synthesized by activated cells.

Unlike the granule-specific components discussed above, these “late stage” mediators are not released immediately following activation of the FcεRI signaling cascade. Accordingly, when quantifying these late stage mediators, care should be taken to insure that the activated cell culture is incubated for a time sufficient to result in the synthesis (if necessary) and release of the mediator being quantified. Generally, PAF and lipid mediators such as leukotriene C4 are released 3-30 min. following FcεRI activation. The cytokines and other late stage mediators are released approx. 4-8 hrs. following FcεRI activation. Incubation times suitable for a specific mediator will be apparent to those of skill in the art. Specific guidance and assays are provided in the Examples section.

The amount of a particular late stage mediator released may be quantified using any standard technique. In one embodiment, the amount(s) may be quantified using ELISA assays. ELISA assay kits suitable for quantifying the amount of TNFα, IL-4, IL-5, IL-6 and/or IL-13 released are available from, for example, Biosource International, Inc., Camarillo, Calif. 93012 (see, e.g., Catalog Nos. KHC3011, KHC0042, KHC0052, KHC0061 and KHC0132). ELISA assay kits suitable for quantifying the amount of leukotriene C4 (LTC4) released from cells are available from Cayman Chemical Co., Ann Arbor, Mich. 48108 (see, e.g., Catalog No. 520211).

Typically, active 2,4-pyrimidinediamine compounds of the invention will exhibit IC₅₀s with respect to FcεRI-mediated degranulation and/or mediator release or synthesis of about 20 μM or lower, as measured in an in vitro assay, such as one of the in vitro assays described above or in the Examples section. Of course, skilled artisans will appreciate that compounds which exhibit lower IC₅₀s, for example on the order of 10 μM, 1 μM, 100 nM, 10 nM, 1 nM, or even lower, are particularly useful.

Skilled artisans will also appreciate that the various mediators discussed above may induce different adverse effects or exhibit different potencies with respect to the same adverse effect. For example, the lipid mediator LTC4 is a potent vasoconstrictor—it is approximately 1000-fold more potent at inducing vasoconstriction than histamine. As another example, in addition to mediating atopic or Type I hypersensitivity reactions, cytokines can also cause tissue remodeling and cell proliferation. Thus, although compounds that inhibit release and/or synthesis of any one of the previously discussed chemical mediators are useful, skilled artisans will appreciate that compounds which inhibit the release and/or synthesis of a plurality, or even all, of the previously described mediators find particular use, as such compounds are useful for ameliorating or avoiding altogether a plurality, or even all, of the adverse effects induced by the particular mediators. For example, compounds which inhibit the release of all three types of mediators—granule-specific, lipid and cytokine—are useful for treating or preventing immediate Type I hypersensitivity reactions as well as the chronic symptoms associated therewith.

Compounds of the invention capable of inhibiting the release of more than one type of mediator (e.g., granule-specific or late stage) may be identified by determining the IC₅₀ with respect to a mediator representative of each class using the various in vitro assays described above (or other equivalent in vitro assays). Compounds of the invention which are capable of inhibiting the release of more than one mediator type will typically exhibit an IC₅₀ for each mediator type tested of less than about 20 μM. For example, a compound which exhibits an IC₅₀ of 1 μM with respect to histamine release (IC₅₀ ^(histamine)) and an IC₅₀ of 1 nM with respect to leukotriene LTC4 synthesis and/or release (IC₅₀ ^(LTC4)) inhibits both immediate (granule-specific) and late stage mediator release. As another specific example, a compound that exhibits an IC₅₀ ^(tryptase) of 10 μM, an IC₅₀ ^(LTC4) of 1 μM and an IC₅₀ ^(IL-4) of 1 μM inhibits immediate (granule-specific), lipid and cytokine mediator release. Although the above specific examples utilize the IC₅₀s of one representative mediator of each class, skilled artisans will appreciate that the IC₅₀s of a plurality, or even all, mediators comprising one or more of the classes may be obtained. The quantity(ies) and identity(ies) of mediators for which IC₅₀ data should be ascertained for a particular compound and application will be apparent to those of skill in the art.

Similar assays may be utilized to confirm inhibition of signal transduction cascades initiated by other Fc receptors, such as FcαRI, FcγRI and/or FcγRIII signaling, with routine modification. For example, the ability of the compounds to inhibit FcγRI signal transduction may be confirmed in assays similar to those described above, with the exception that the FcγRI signaling cascade is activated, for example by incubating the cells with IgG and an IgG-specific allergen or antibody, instead of IgE and an IgE-specific allergen or antibody. Suitable cell types, activating agents and agents to quantify to confirm inhibition of other Fc receptors, such as Fc receptors that comprise a gamma homodimer, will be apparent to those of skill in the art.

One particularly useful class of compounds includes those 2,4-pyrimidinediamine compounds that inhibit the release of immediate granule-specific mediators and late stage mediators with approximately equivalent IC₅₀s. By approximately equivalent is meant that the IC₅₀s for each mediator type are within about a 10-fold range of one another. Another particularly useful class of compounds includes those 2,4-pyrimidinediamine compounds that inhibit the release of immediate granule-specific mediators, lipid mediators and cytokine mediators with approximately equivalent IC₅₀s. In a specific embodiment, such compounds inhibit the release of the following mediators with approximately equivalent IC₅₀s: histamine, tryptase, hexosaminidase, IL-4, IL-5, IL-6, IL-13, TNFα and LTC4. Such compounds are particularly useful for, among other things, ameliorating or avoiding altogether both the early and late stage responses associated with atopic or immediate Type I hypersensitivity reactions.

Ideally, the ability to inhibit the release of all desired types of mediators will reside in a single compound. However, mixtures of compounds can also be identified that achieve the same result. For example, a first compound which inhibits the release of granule specific mediators may be used in combination with a second compound which inhibits the release and/or synthesis of cytokine mediators.

In addition to the FcεRI or FcγRI degranulation pathways discussed above, degranulation of mast and/or basophil cells can be induced by other agents. For example, ionomycin, a calcium ionophore that bypasses the early FcεRI or FcγRI signal transduction machinery of the cell, directly induces a calcium flux that triggers degranulation. Referring again to FIG. 2, activated PLCγ initiates pathways that lead to, among other things, calcium ion mobilization and subsequent degranulation. As illustrated, this Ca²⁺ mobilization is triggered late in the FcεRI signal transduction pathway. As mentioned above, and as illustrated in FIG. 3, ionomycin directly induces Ca²⁺ mobilization and a Ca²⁺ flux that leads to degranulation. Other ionophores that induce degranulation in this manner include A23187. The ability of granulation-inducing ionophores such as ionomycin to bypass the early stages of the FcεRI and/or FcγRI signaling cascades may be used as a counter screen to identify active compounds of the invention that specifically exert their degranulation-inhibitory activity by blocking or inhibiting the early FcεRI or FcγRI signaling cascades, as discussed above. Compounds which specifically inhibit such early FcεRI or FcγRI-mediated degranulation inhibit not only degranulation and subsequent rapid release of histamine, tryptase and other granule contents, but also inhibit the pro-inflammatory activation pathways causing the release of TNFα, IL-4, IL-13 and the lipid mediators such as LTC4. Thus, compounds which specifically inhibit such early FcεRI and/or FcγRI -mediated degranulation block or inhibit not only acute atopic or Type I hypersensitivity reactions, but also late responses involving multiple inflammatory mediators.

Compounds of the invention that specifically inhibit early FcεRI and/or FcγRI-mediated degranulation are those compounds that inhibit FcεRI and/or FcγRI-mediated degranulation (for example, have an IC₅₀ of less than about 20 μM with respect to the release of a granule-specific mediator or component as measured in an in vitro assay with cells stimulated with an IgE or IgG binding agent) but that do not appreciably inhibit ionophore-induced degranulation. In one embodiment, compounds are considered to not appreciably inhibit ionophore-induced degranulation if they exhibit an IC₅₀ of ionophore-induced degranulation of greater than about 20 μM, as measured in an in vitro assay. Of course, active compounds that exhibit even higher IC₅₀s of ionophore-induced degranulation, or that do not inhibit ionophore-induced degranulation at all, are particularly useful. In another embodiment, compounds are considered to not appreciably inhibit ionophore-induced degranulation if they exhibit a greater than 10-fold difference in their IC₅₀s of FcεRI and/or FcγRI-mediated degranulation and ionophore-induced degranulation, as measured in an in vitro assay. Assays suitable for determining the IC₅₀ of ionophore-induced degranulation include any of the previously-described degranulation assays, with the modification that the cells are stimulated or activated with a degranulation-inducing calcium ionophore such as ionomycin or A23187 (A.G. Scientific, San Diego, Calif.) instead of anti-IgE antibodies or an IgE-specific allergen. Specific assays for assessing the ability of a particular 2,4-pyrimidinediamine compound of the invention to inhibit ionophore-induced degranulation are provided in the Examples section.

As will be recognized by skilled artisans, compounds which exhibit a high degree of selectivity of FcεRI-mediated degranulation find particular use, as such compounds selectively target the FcεRI cascade and do not interfere with other degranulation mechanisms. Similarly, compounds which exhibit a high degree of selectivity of FcγRI-mediated degranulation find particular use, as such compounds selectively target the FcγRI cascade and do not interfere with other degranulation mechanisms. Compounds which exhibit a high degree of selectivity are generally 10-fold or more selective for FcεRI- or FcγRI-mediated degranulation over ionophore-induced degranulation, such as ionomycin-induced degranulation.

Biochemical and other data confirm that the 2,4-pyrimidinediamine compounds described herein are potent inhibitors of Syk kinase activity. For example, in experiments with an isolated Syk kinase, of twenty four 2,4-pyrimidinediamine compounds tested, all but two inhibited the Syk kinase catalyzed phosphorylation of a peptide substrate with IC50s in the submicromolar range. The remaining compounds inhibited phosphorylation in the micromolar range. In addition, of sixteen compounds tested in an in vitro assay with mast cells, all inhibited phosphorylation of Syk kinase substrates (e.g., PLC-gamma1, LAT) and proteins downstream of Syk kinase (e.g., JNK, p38, Erk1/2 and PKB, when tested), but not proteins upstream of Syk kinase in the cascade (e.g., Lyn). Phosphorylation of Lyn substrates was not inhibited by the 2,4-pyrimidinediamine compounds tested. Moreover, for the following compounds, a high correlation was observed between their inhibition of Syk kinase activity in biochemical assays (IC₅₀s in the range of 3 to 1850 nM) and their inhibition of FcεR1-mediated degranulation in mast cells (IC₅₀s in the range of 30 to 1650 nM): R950373, R950368, R921302, R945371, R945370, R945369, R945365, R921304, R945144, R945140, R945071, R940358, R940353, R940352, R940351, R940350, R940347, R921303, R940338, R940323, R940290, R940277, R940276, R940275, R940269, R940255, R935393, R935372, R935366, R935310, R935309, R935307, R935304, R935302, R935293, R935237, R935198, R935196, R935194, R935193, R935191, R935190, R935138, R927050, R926968, R926956, R926931, R926891, R926839, R926834, R926816, R926813, R926791, R926782, R926780, R926757, R926753, R926745, R926715, R926508, R926505, R926502, R926501, R926500, R921218, R921147, R920410, R909268, R921219, R908712, R908702.

Accordingly, the activity of the 2,4-pyrimidinediamine compounds of the invention may also be confirmed in biochemical or cellular assays of Syk kinase activity. Referring again to FIG. 2, in the FcεRI signaling cascade in mast and/or basophil cells, Syk kinase phosphorylates LAT and PLC-gamma1, which leads to, among other things, degranulation. Any of these activities may be used to confirm the activity of the 2,4-pyrimidinediamine compounds of the invention. In one embodiment, the activity is confirmed by contacting an isolated Syk kinase, or an active fragment thereof with a 2,4-pyrimidinediamine compound in the presence of a Syk kinase substrate (e.g., a synthetic peptide or a protein that is known to be phosphorylated by Syk in a signaling cascade) and assessing whether the Syk kinase phosphorylated the substrate. Alternatively, the assay may be carried out with cells that express a Syk kinase. The cells may express the Syk kinase endogenously or they may be engineered to express a recombinant Syk kinase. The cells may optionally also express the Syk kinase substrate. Cells suitable for performing such confirmation assays, as well as methods of engineering suitable cells will be apparent to those of skill in the art. Specific examples of biochemical and cellular assays suitable for confirming the activity of the 2,4-pyrimidinediamine compounds are provided in the Examples section.

Generally, compounds that are Syk kinase inhibitors will exhibit an IC₅₀ with respect to a Syk kinase activity, such as the ability of Syk kinase to phosphorylate a synthetic or endogenous substrate, in an in vitro or cellular assay in the range of about 20 μM or less. Skilled artisans will appreciate that compounds that exhibit lower IC50s, such as in the range of 10 μM, 1 μM, 100 nM, 10 nM, 1 nM, or even lower, are particularly useful.

6.5 Uses and Compositions

As previously discussed, the active compounds of the invention inhibit Fc receptor signaling cascades, especially those Fc receptors including a gamma homodimer, such as the FcεRI and/or FcγRI signaling cascades, that lead to, among other things, the release and/or synthesis of chemical mediators from cells, either via degranulation or other processes. As also discussed, the active compounds are also potent inhibitors of Syk kinase. As a consequence of these activities, the active compounds of the invention may be used in a variety of in vitro, in vivo and ex vivo contexts to regulate or inhibit Syk kinase, signaling cascades in which Syk kinase plays a role, Fc receptor signaling cascades, and the biological responses effected by such signaling cascades. For example, in one embodiment, the compounds may be used to inhibit Syk kinase, either in vitro or in vivo, in virtually any cell type expressing Syk kinase. They may also be used to regulate signal transduction cascades in which Syk kinase plays a role. Such Syk-dependent signal transduction cascades include, but are not limited to, the FcεR1, FcγRI, FcγRIII, BCR and integrin signal transduction cascades. The compounds may also be used in vitro or in vivo to regulate, and in particular inhibit, cellular or biological responses effected by such Syk-dependent signal transduction cascades. Such cellular or biological responses include, but are not limited to, respiratory burst, cellular adhesion, cellular degranulation, cell spreading, cell migration, cell aggregation, phagcytosis, cytokine synthesis and release, cell maturation and Ca²⁺ flux. Importantly, the compounds may be used to inhibit Syk kinase in vivo as a therapeutic approach towards the treatment or prevention of diseases mediated, either wholly or in part, by a Syk kinase activity. Non-limiting examples of Syk kinase mediated diseases that may be treated or prevented with the compounds are those discussed in more detail, below.

In another embodiment, the active compounds may be used to regulate or inhibit the Fc receptor signaling cascades and/or FcεRI- and/or FcγRI-mediated degranulation as a therapeutic approach towards the treatment or prevention of diseases characterized by, caused by and/or associated with the release or synthesis of chemical mediators of such Fc receptor signaling cascades or degranulation. Such treatments may be administered to animals in veterinary contexts or to humans. Diseases that are characterized by, caused by or associated with such mediator release, synthesis or degranulation, and that can therefore be treated or prevented with the active compounds include, by way of example and not limitation, atopy or anaphylactic hypersensitivity or allergic reactions, allergies (e.g., allergic conjunctivitis, allergic rhinitis, atopic asthma, atopic dermatitis and food allergies), low grade scarring (e.g., of scleroderma, increased fibrosis, keloids, post-surgical scars, pulmonary fibrosis, vascular spasms, migraine, reperfusion injury and post myocardial infarction), diseases associated with tissue destruction (e.g., of COPD, cardiobronchitis and post myocardial infarction), diseases associated with tissue inflammation (e.g., irritable bowel syndrome, spastic colon and inflammatory bowel disease), inflammation and scarring.

In addition to the myriad diseases discussed above, cellular and animal empirical data confirm that the 2,4-pyrimidinediamine compounds described herein are also useful for the treatment or prevention of autoimmune diseases, as well as the various symptoms associated with such diseases. The types of autoimmune diseases that may be treated or prevented with the 2,4-pyrimidinediamine compounds generally include those disorders involving tissue injury that occurs as a result of a humoral and/or cell-mediated response to immunogens or antigens of endogenous and/or exogenous origin. Such diseases are frequently referred to as diseases involving the nonanaphylactic (i.e., Type II, Type III and/or Type IV) hypersensitivity reactions.

As discussed previously, Type I hypersensitivity reactions generally result from the release of pharmacologically active substances, such as histamine, from mast and/or basophil cells following contact with a specific exogenous antigen. As mentioned above, such Type I reactions play a role in numerous diseases, including allergic asthma, allergic rhinitis, etc.

Type II hypersensitivity reactions (also referred to as cytotoxic, cytolytic complement-dependent or cell-stimulating hypersensitivity reactions) result when immunoglobulins react with antigenic components of cells or tissue, or with an antigen or hapten that has become intimately coupled to cells or tissue. Diseases that are commonly associated with Type II hypersensitivity reactions include, but are not limited, to autoimmune hemolytic anemia, erythroblastosis fetalis and Goodpasture's disease.

Type III hypersensitivity reactions, (also referred to as toxic complex, soluble complex, or immune complex hypersensitivity reactions) result from the deposition of soluble circulating antigen-immunoglobulin complexes in vessels or in tissues, with accompanying acute inflammatory reactions at the site of immune complex deposition. Non-limiting examples of prototypical Type III reaction diseases include the Arthus reaction, rheumatoid arthritis, serum sickness, systemic lupus erythematosis, certain types of glomerulonephritis, multiple sclerosis and bullous pemphingoid.

Type IV hypersensitivity reactions (frequently called cellular, cell-mediated, delayed, or tuberculin-type hypersensitivity reactions) are caused by sensitized T-lymphocytes which result from contact with a specific antigen. Non-limiting examples of diseases cited as involving Type IV reactions are contact dermatitis and allograft rejection.

Autoimmune diseases associated with any of the above nonanaphylactic hypersensitivity reactions may be treated or prevented with the 2,4-pyrimidinediamine compounds of the invention. In particular, the methods may be used to treat or prevent those autoimmune diseases frequently characterized as single organ or single cell-type autoimmune disorders including, but not limited to: Hashimoto's thyroiditis, autoimmune hemolytic anemia, autoimmune atrophic gastritis of pernicious anemia, autoimmune encephalomyelitis, autoimmune orchitis, Goodpasture's disease, autoimmune thrombocytopenia, sympathetic ophthalmia, myasthenia gravis, Graves' disease, primary biliary cirrhosis, chronic aggressive hepatitis, ulcerative colitis and membranous glomerulopathy, as well as those autoimmune diseases frequently characterized as involving systemic autoimmune disorder, which include but are not limited to: systemic lupus erythematosis, rheumatoid arthritis, Sjogren's syndrome, Reiter's syndrome, polymyositis-dermatomyositis, systemic sclerosis, polyarteritis nodosa, multiple sclerosis and bullous pemphigoid.

It will be appreciated by skilled artisans that many of the above-listed autoimmune diseases are associated with severe symptoms, the amelioration of which provides significant therapeutic benefit even in instances where the underlying autoimmune disease may not be ameliorated. Many of these symptoms, as well as their underlying disease states, result as a consequence of activating the FcγR signaling cascade in monocyte cells. As the 2,4-pyrimidinediamine compounds described herein are potent inhibitors of such FcγR signaling in monocytes and other cells, the methods find use in the treatment and/or prevention of myriad adverse symptoms associated with the above-listed autoimmune diseases.

As a specific example, rheumatoid arthritis (RA) typically results in swelling, pain, loss of motion and tenderness of target joints throughout the body. RA is characterized by chronically inflamed synovium that is densely crowded with lymphocytes. The synovial membrane, which is typically one cell layer thick, becomes intensely cellular and assumes a form similar to lymphoid tissue, including dentritic cells, T-, B- and NK cells, macrophages and clusters of plasma cells. This process, as well as a plethora of immunopathological mechanisms including the formation of antigen-immunoglobulin complexes, eventually result in destruction of the integrity of the joint, resulting in deformity, permanent loss of function and/or bone erosion at or near the joint. The methods may be used to treat or ameliorate any one, several or all of these symptoms of RA. Thus, in the context of RA, the methods are considered to provide therapeutic benefit (discussed more generally, infra) when a reduction or amelioration of any of the symptoms commonly associated with RA is achieved, regardless of whether the treatment results in a concomitant treatment of the underlying RA and/or a reduction in the amount of circulating rheumatoid factor (“RF”).

As another specific example, systemic lupus erythematosis (“SLE”) is typically associated with symptoms such as fever, joint pain (arthralgias), arthritis, and serositis (pleurisy or pericarditis). In the context of SLE, the methods are considered to provide therapeutic benefit when a reduction or amelioration of any of the symptoms commonly associated with SLE are achieved, regardless of whether the treatment results in a concomitant treatment of the underlying SLE.

As another specific example, multiple sclerosis (“MS”) cripples the patient by disturbing visual acuity; stimulating double vision; disturbing motor functions affecting walking and use of the hands; producing bowel and bladder incontinence; spasticity; and sensory deficits (touch, pain and temperature sensitivity). In the context of MS, the methods are considered to provide therapeutic benefit when an improvement or a reduction in the progression of any one or more of the crippling effects commonly associated with MS is achieved, regardless of whether the treatment results in a concomitant treatment of the underlying MS.

When used to treat or prevent such diseases, the active compounds may be administered singly, as mixtures of one or more active compounds or in mixture or combination with other agents useful for treating such diseases and/or the symptoms associated with such diseases. The active compounds may also be administered in mixture or in combination with agents useful to treat other disorders or maladies, such as steroids, membrane stabilizers, 5LO inhibitors, leukotriene synthesis and receptor inhibitors, inhibitors of IgE isotype switching or IgE synthesis, IgG isotype switching or IgG synthesis, β-agonists, tryptase inhibitors, aspirin, COX inhibitors, methotrexate, anti-TNF drugs, retuxin, PD4 inhibitors, p38 inhibitors, PDE4 inhibitors, and antihistamines, to name a few. The active compounds may be administered per se in the form of prodrugs or as pharmaceutical compositions, comprising an active compound or prodrug.

Pharmaceutical compositions comprising the active compounds of the invention (or prodrugs thereof) may be manufactured by means of conventional mixing, dissolving, granulating, dragee-making levigating, emulsifying, encapsulating, entrapping or lyophilization processes. The compositions may be formulated in conventional manner using one or more physiologically acceptable carriers, diluents, excipients or auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically.

The active compound or prodrug may be formulated in the pharmaceutical compositions per se, or in the form of a hydrate, solvate, N-oxide or pharmaceutically acceptable salt, as previously described. Typically, such salts are more soluble in aqueous solutions than the corresponding free acids and bases, but salts having lower solubility than the corresponding free acids and bases may also be formed.

Pharmaceutical compositions of the invention may take a form suitable for virtually any mode of administration, including, for example, topical, ocular, oral, buccal, systemic, nasal, injection, transdermal, rectal, vaginal, etc., or a form suitable for administration by inhalation or insufflation.

For topical administration, the active compound(s) or prodrug(s) may be formulated as solutions, gels, ointments, creams, suspensions, etc. as are well-known in the art.

Systemic formulations include those designed for administration by injection, e.g., subcutaneous, intravenous, intramuscular, intrathecal or intraperitoneal injection, as well as those designed for transdermal, transmucosal oral or pulmonary administration.

Useful injectable preparations include sterile suspensions, solutions or emulsions of the active compound(s) in aqueous or oily vehicles. The compositions may also contain formulating agents, such as suspending, stabilizing and/or dispersing agent. The formulations for injection may be presented in unit dosage form, e.g., in ampules or in multidose containers, and may contain added preservatives.

Alternatively, the injectable formulation may be provided in powder form for reconstitution with a suitable vehicle, including but not limited to sterile pyrogen free water, buffer, dextrose solution, etc., before use. To this end, the active compound(s) may be dried by any art-known technique, such as lyophilization, and reconstituted prior to use.

For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are known in the art.

For oral administration, the pharmaceutical compositions may take the form of, for example, lozenges, tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., pregelatinised maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc or silica); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulfate). The tablets may be coated by methods well known in the art with, for example, sugars, films or enteric coatings. Compounds which are particularly suitable for oral administration include Compounds R940350, R935372, R935193, R927050 and R935391.

Liquid preparations for oral administration may take the form of, for example, elixirs, solutions, syrups or suspensions, or they may be presented as a dry product for constitution with water or other suitable vehicle before use. Such liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats);

emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters, ethyl alcohol, Cremophore™ or fractionated vegetable oils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid). The preparations may also contain buffer salts, preservatives, flavoring, coloring and sweetening agents as appropriate.

Preparations for oral administration may be suitably formulated to give controlled release of the active compound or prodrug, as is well known.

For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner.

For rectal and vaginal routes of administration, the active compound(s) may be formulated as solutions (for retention enemas) suppositories or ointments containing conventional suppository bases such as cocoa butter or other glycerides.

For nasal administration or administration by inhalation or insufflation, the active compound(s) or prodrug(s) can be conveniently delivered in the form of an aerosol spray from pressurized packs or a nebulizer with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, fluorocarbons, carbon dioxide or other suitable gas. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges for use in an inhaler or insufflator (for example capsules and cartridges comprised of gelatin) may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.

A specific example of an aqueous suspension formulation suitable for nasal administration using commercially-available nasal spray devices includes the following ingredients: active compound or prodrug (0.5-20 mg/ml); benzalkonium chloride (0.1-0.2 mg/mL); polysorbate 80 (TWEEN® 80; 0.5-5 mg/ml); carboxymethylcellulose sodium or microcrystalline cellulose (1-15 mg/ml); phenylethanol (1-4 mg/ml); and dextrose (20-50 mg/ml). The pH of the final suspension can be adjusted to range from about pH5 to pH7, with a pH of about pH 5.5 being typical.

Another specific example of an aqueous suspension suitable for administration of the compounds via inhalation, and in particular for such administration of Compound R921218, contains 1-20 mg/mL Compound or prodrug, 0.1-1% (v/v) Polysorbate 80 (TWEEN®80), 50 mM citrate and/or 0.9% sodium chloride.

For ocular administration, the active compound(s) or prodrug(s) may be formulated as a solution, emulsion, suspension, etc. suitable for administration to the eye. A variety of vehicles suitable for administering compounds to the eye are known in the art. Specific non-limiting examples are described in U.S. Pat. No. 6,261,547; U.S. Pat. No. 6,197,934; U.S. Pat. No. 6,056,950; U.S. Pat. No. 5,800,807; U.S. Pat. No. 5,776,445; U.S. Pat. No. 5,698,219; U.S. Pat. No. 5,521,222; U.S. Pat. No. 5,403,841; U.S. Pat. No. 5,077,033; U.S. Pat. No. 4,882,150; and U.S. Pat. No. 4,738,851.

For prolonged delivery, the active compound(s) or prodrug(s) can be formulated as a depot preparation for administration by implantation or intramuscular injection. The active ingredient may be formulated with suitable polymeric or hydrophobic materials (e.g., as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, e.g., as a sparingly soluble salt. Alternatively, transdermal delivery systems manufactured as an adhesive disc or patch which slowly releases the active compound(s) for percutaneous absorption may be used. To this end, permeation enhancers may be used to facilitate transdermal penetration of the active compound(s). Suitable transdermal patches are described in for example, U.S. Pat. No. 5,407,713; U.S. Pat. No. 5,352,456; U.S. Pat. No. 5,332,213; U.S. Pat. No. 5,336,168; U.S. Pat. No. 5,290,561; U.S. Pat. No. 5,254,346; U.S. Pat. No. 5,164,189; U.S. Pat. No. 5,163,899; U.S. Pat. No. 5,088,977; U.S. Pat. No. 5,087,240; U.S. Pat. No. 5,008,110; and U.S. Pat. No. 4,921,475.

Alternatively, other pharmaceutical delivery systems may be employed. Liposomes and emulsions are well-known examples of delivery vehicles that may be used to deliver active compound(s) or prodrug(s). Certain organic solvents such as dimethylsulfoxide (DMSO) may also be employed, although usually at the cost of greater toxicity.

The pharmaceutical compositions may, if desired, be presented in a pack or dispenser device which may contain one or more unit dosage forms containing the active compound(s). The pack may, for example, comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration.

6.6 Effective Dosages

The active compound(s) or prodrug(s) of the invention, or compositions thereof, will generally be used in an amount effective to achieve the intended result, for example in an amount effective to treat or prevent the particular disease being treated. The compound(s) may be administered therapeutically to achieve therapeutic benefit or prophylactically to achieve prophylactic benefit. By therapeutic benefit is meant eradication or amelioration of the underlying disorder being treated and/or eradication or amelioration of one or more of the symptoms associated with the underlying disorder such that the patient reports an improvement in feeling or condition, notwithstanding that the patient may still be afflicted with the underlying disorder. For example, administration of a compound to a patient suffering from an allergy provides therapeutic benefit not only when the underlying allergic response is eradicated or ameliorated, but also when the patient reports a decrease in the severity or duration of the symptoms associated with the allergy following exposure to the allergen. As another example, therapeutic benefit in the context of asthma includes an improvement in respiration following the onset of an asthmatic attack, or a reduction in the frequency or severity of asthmatic episodes. Therapeutic benefit also includes halting or slowing the progression of the disease, regardless of whether improvement is realized.

For prophylactic administration, the compound may be administered to a patient at risk of developing one of the previously described diseases. For example, if it is unknown whether a patient is allergic to a particular drug, the compound may be administered prior to administration of the drug to avoid or ameliorate an allergic response to the drug. Alternatively, prophylactic administration may be applied to avoid the onset of symptoms in a patient diagnosed with the underlying disorder. For example, a compound may be administered to an allergy sufferer prior to expected exposure to the allergen. Compounds may also be administered prophylactically to healthy individuals who are repeatedly exposed to agents known to one of the above-described maladies to prevent the onset of the disorder. For example, a compound may be administered to a healthy individual who is repeatedly exposed to an allergen known to induce allergies, such as latex, in an effort to prevent the individual from developing an allergy. Alternatively, a compound may be administered to a patient suffering from asthma prior to partaking in activities which trigger asthma attacks to lessen the severity of, or avoid altogether, an asthmatic episode.

The amount of compound administered will depend upon a variety of factors, including, for example, the particular indication being treated, the mode of administration, whether the desired benefit is prophylactic or therapeutic, the severity of the indication being treated and the age and weight of the patient, the bioavailability of the particular active compound, etc. Determination of an effective dosage is well within the capabilities of those skilled in the art.

Effective dosages may be estimated initially from in vitro assays. For example, an initial dosage for use in animals may be formulated to achieve a circulating blood or serum concentration of active compound that is at or above an IC₅₀ of the particular compound as measured in as in vitro assay, such as the in vitro CHMC or BMMC and other in vitro assays described in the Examples section. Calculating dosages to achieve such circulating blood or serum concentrations taking into account the bioavailability of the particular compound is well within the capabilities of skilled artisans. For guidance, the reader is referred to Fingl & Woodbury, “General Principles,” In: Goodman and Gilman's The Pharmaceutical Basis of Therapeutics, Chapter 1, pp. 1-46, latest edition, Pagamonon Press, and the references cited therein.

Initial dosages can also be estimated from in vivo data, such as animal models. Animal models useful for testing the efficacy of compounds to treat or prevent the various diseases described above are well-known in the art. Suitable animal models of hypersensitivity or allergic reactions are described in Foster, 1995, Allergy 50(21Suppl):6-9, discussion 34-38 and Tumas et al., 2001, J. Allergy Clin. Immunol. 107(6):1025-1033. Suitable animal models of allergic rhinitis are described in Szelenyi et al., 2000, Arzneimittelforschung 50(11):1037-42; Kawaguchi et al., 1994, Clin. Exp. Allergy 24(3):238-244 and Sugimoto et al., 2000, Immunopharmacology 48(1):1-7. Suitable animal models of allergic conjunctivitis are described in Carreras et al., 1993, Br. J. Ophthalmol. 77(8):509-514; Saiga et al., 1992, Ophthalmic Res. 24(1):45-50; and Kunert et al., 2001, Invest. Ophthalmol. Vis. Sci. 42(11):2483-2489. Suitable animal models of systemic mastocytosis are described in O'Keefe et al., 1987, J. Vet. Intern. Med. 1(2):75-80 and Bean-Knudsen et al., 1989, Vet. Pathol. 26(1):90-92. Suitable animal models of hyper IgE syndrome are described in Claman et al., 1990, Clin. Immunol. Immunopathol. 56(1):46-53. Suitable animal models of B-cell lymphoma are described in Hough et al., 1998, Proc. Natl. Acad. Sci. USA 95:13853-13858 and Hakim et al., 1996, J. Immunol. 157(12):5503-5511. Suitable animal models of atopic disorders such as atopic dermatitis, atopic eczema and atopic asthma are described in Chan et al., 2001, J. Invest. Dermatol. 117(4):977-983 and Suto et al., 1999, Int. Arch. Allergy Immunol. 120(Suppl 1):70-75. Ordinarily skilled artisans can routinely adapt such information to determine dosages suitable for human administration. Additional suitable animal models are described in the Examples section.

Dosage amounts will typically be in the range of from about 0.0001 or 0.001 or 0.01 mg/kg/day to about 100 mg/kg/day, but may be higher or lower, depending upon, among other factors, the activity of the compound, its bioavailability, the mode of administration and various factors discussed above. Dosage amount and interval may be adjusted individually to provide plasma levels of the compound(s) which are sufficient to maintain therapeutic or prophylactic effect. For example, the compounds may be administered once per week, several times per week (e.g., every other day), once per day or multiple times per day, depending upon, among other things, the mode of administration, the specific indication being treated and the judgment of the prescribing physician. In cases of local administration or selective uptake, such as local topical administration, the effective local concentration of active compound(s) may not be related to plasma concentration. Skilled artisans will be able to optimize effective local dosages without undue experimentation.

Preferably, the compound(s) will provide therapeutic or prophylactic benefit without causing substantial toxicity. Toxicity of the compound(s) may be determined using standard pharmaceutical procedures. The dose ratio between toxic and therapeutic (or prophylactic) effect is the therapeutic index. Compounds(s) that exhibit high therapeutic indices are preferred.

The invention having been described, the following examples are offered by way of illustration and not limitation.

7. EXAMPLES 7.1 Synthesis of Starting Materials and Intermediates Useful for Synthesizing the 2,4-Pyrimidinediamine Compounds According to Schemes (I)-(V)

A variety of starting materials and N4-monosubstituted-2-pyrimidineamines and N2-monosubstituted-4-pyrimidinediamines [mono Substitution Nucleophilic Aromatic Reaction (SNAR) products] useful for synthesizing the 2,4-pyrimidinediamine compounds of the invention according to Schemes (I)-(V) were prepared as described below. Conditions suitable for synthesizing the mono SNAR products are exemplified with 2-chloro-N4-(3,4-ethylenedioxyphenyl)-5-fluoro-4-pyrimidineamine (R926087).

Lengthy table referenced here US20120230984A1-20120913-T00001 Please refer to the end of the specification for access instructions.

7.5 The 2,4-Pyrimidinediamine Compounds of the Invention Inhibit FcεRI Receptor-Mediated Degranulation

The ability of the 2,4-pyrimidinediamine compounds of the invention to inhibit IgE-induced degranulation was demonstrated in a variety of cellular assays with cultured human mast cells (CHMC) and/or mouse bone marrow derived cells (BMMC). Inhibition of degranulation was measured at both low and high cell density by quantifying the release of the granule specific factors tryptase, histamine and hexosaminidase. Inhibition of release and/or synthesis of lipid mediators was assessed by measuring the release of leukotriene LTC4 and inhibition of release and/or synthesis of cytokines was monitored by quantifying TNF-α, IL-6 and IL-13. Tryptase and hexosaminidase were quantified using fluorogenic substrates as described in their respective examples. Histamine, TNFα, IL-6, IL-13 and LTC4 were quantified using the following commercial ELISA kits: histamine (Immunotech #2015, Beckman Coulter), TNFα (Biosource #KHC3011), IL-6 (Biosource #KMC0061), IL-13 (Biosource #KHC0132) and LTC4 (Cayman Chemical #520211). The protocols of the various assays are provided below.

7.5.1 Culturing of Human Mast and Basophil Cells

Human mast and basophil cells were cultured from CD34-negative progenitor cells as described below (see also the methods described in copending U.S. application Ser. No. 10/053,355, filed Nov. 8, 2001, the disclosure of which is incorporated herein by reference).

7.5.1.1 Preparation of STEMPRO-34 Complete Medium

To prepare STEMPRO-34 complete medium (“CM”), 250 mL STEMPRO-34™ serum free medium (“SFM”; GibcoBRL, Catalog No. 10640) was added to a filter flask. To this was added 13 mL STEMPRO-34 Nutrient Supplement (“NS”; GibcoBRL, Catalog No. 10641) (prepared as described in more detail, below). The NS container was rinsed with approximately 10 mL SFM and the rinse added to the filter flask. Following addition of 5 mL L-glutamine (200 mM; Mediatech, Catalog No. MT 25-005-CI and 5 mL 100× penicillin/streptomycin (“pen-strep”; HyClone, Catalog No. SV30010), the volume was brought to 500 mL with SFM and the solution was filtered.

The most variable aspect of preparing the CM is the method by which the NS is thawed and mixed prior to addition to the SFM. The NS should be thawed in a 37° C. water bath and swirled, not vortexed or shaken, until it is completely in solution. While swirling, take note whether there are any lipids that are not yet in solution. If lipids are present and the NS is not uniform in appearance, return it to the water bath and repeat the swirling process until it is uniform in appearance. Sometimes this component goes into solution immediately, sometimes after a couple of swirling cycles, and sometimes not at all. If, after a couple of hours, the NS is still not in solution, discard it and thaw a fresh unit. NS that appears non-uniform after thaw should not be used.

7.5.1.2 Expansion of CD34+ Cells

A starting population of CD34-positive (CD34+) cells of relatively small number (1-5×10⁶ cells) was expanded to a relatively large number of CD34-negative progenitor cells (about 2-4×10⁹ cells) using the culture media and methods described below. The CD34+ cells (from a single donor) were obtained from Allcells (Berkeley, Calif.). Because there is a degree of variation in the quality and number of CD34+ cells that Allcells typically provides, the newly delivered cells were transferred to a 15 mL conical tube and brought up to 10 mL in CM prior to use.

On day 0, a cell count was performed on the viable (phase-bright) cells and the cells were spun at 1200 rpm to pellet. The cells were resuspended to a density of 275,000 cells/mL with CM containing 200 ng/mL recombinant human Stem Cell Factor (“SCF”; Peprotech, Catalog No. 300-07) and 20 ng/mL human flt-3 ligand (Peprotech, Catalog No. 300-19) (“CM/SCF/flt-3 medium”). On about day 4 or 5, the density of the culture was checked by performing a cell count and the culture was diluted to a density of 275,000 cells/mL with fresh CM/SCF/flt-3 medium. On about day 7, the culture was transferred to a sterile tube and a cell count was performed. The cells were spun at 1200 rpm and resuspended to a density of 275,000 cells/mL with fresh CM/SCF/flt-3 medium.

This cycle was repeated, starting from day 0, a total of 3-5 times over the expansion period.

When the culture is large and being maintained in multiple flasks and is to be resuspended, the contents of all of the flasks are combined into a single container prior to performing a cell count. This ensures that an accurate cell count is achieved and provides for a degree of uniformity of treatment for the entire population. Each flask is checked separately for contamination under the microscope prior to combining to prevent contamination of the entire population.

Between days 17-24, the culture can begin to go into decline (i.e., approximately 5-10% of the total number of cells die) and fail to expand as rapidly as before. The cells are then monitored on a daily basis during this time, as complete failure of the culture can take place in as little as 24 hours. Once the decline has begun, the cells are counted, spun down at 850 rpm for 15 minutes, and resuspended at a density of 350,000 cells/mL in CM/SCF/flt-3 medium to induce one or two more divisions out of the culture. The cells are monitored daily to avoid failure of the culture.

When greater than 15% cell death is evident in the progenitor cell culture and some debris is present in the culture, the CD34-negative progenitor cells are ready to be differentiated.

7.5.1.3 Differentiation of CD34-Negative Progenitor Cells into Mucosal Mast Cells

A second phase is performed to convert the expanded CD34-negative progenitor cells into differentiated mucosal mast cells. These mucosal cultured human mast cells (“CHMC”) are derived from CD34+ cells isolated from umbilical cord blood and treated to form a proliferated population of CD34-negative progenitor cells, as described above. To produce the CD43-negative progenitor cells, the resuspension cycle for the culture was the same as that described above, except that the culture was seeded at a density of 425,000 cells/mL and 15% additional media was added on about day four or five without performing a cell count. Also, the cytokine composition of the medium was modified such that it contained SCF (200 ng/mL) and recombinant human IL-6 (200 ng/mL; Peprotech, Catalog No. 200-06 reconstituted to 100 ug/mL in sterile 10 mM acetic acid) (“CM/SCF/IL-6 medium”).

Phases I and II together span approximately 5 weeks. Some death and debris in the culture is evident during weeks 1-3 and there is a period during weeks 2-5 during which a small percentage of the culture is no longer in suspension, but is instead attached to the surface of the culture vessel.

As during Phase I, when the culture is to be resuspended on day seven of each cycle, the contents of all flasks are combined into a single container prior to performing a cell count to ensure uniformity of the entire population. Each flask is checked separately for contamination under the microscope prior to combining to prevent contamination of the entire population.

When the flasks are combined, approximately 75% of the volume is transferred to the communal container, leaving behind about 10 mL or so in the flask. The flask containing the remaining volume was rapped sharply and laterally to dislodge the attached cells. The rapping was repeated at a right angle to the first rap to completely dislodge the cells.

The flask was leaned at a 45 degree angle for a couple of minutes before the remaining volume was transferred to the counting vessel. The cells were spun at 950 rpm for 15 min prior to seeding at 35-50 mL per flask (at a density of 425,000 cells/mL).

7.5.1.4 Differentiation of CD34-Negative Progenitor Cells into Connective Tissue-Type Mast Cells

A proliferated population of CD34-negative progenitor cells is prepared as above and treated to form a tryptase/chymase positive (connective tissue) phenotype. The methods are performed as described above for mucosal mast cells, but with the substitution of IL-4 for IL-6 in the culture medium. The cells obtained are typical of connective tissue mast cells.

7.5.1.5 Differentiation of CD34-Negative Progenitor Cells into Basophil Cells

A proliferated population of CD34-negative progenitor cells is prepared as described in Section 7.5.1.3, above, and used to form a proliferated population of basophil cells. The CD34-negative cells are treated as described for mucosal mast cells, but with the substitution of IL-3 (at 20-50 ng/mL) for IL-6 in the culture medium.

7.5.2 CHMC Low Cell Density IgE Activation Tryptase and LTC4 Assays

To duplicate 96-well U-bottom plates (Costar 3799) add 65 ul of compound dilutions or control samples that have been prepared in MT [137 mM NaCl, 2.7 mM KCl, 1.8 mM CaCl₂, 1.0 mM MgCl₂, 5.6 mM Glucose, 20 mM Hepes (pH 7.4), 0.1% Bovine Serum Albumin, (Sigma A4503)] containing 2% MeOH and 1% DMSO. Pellet CHMC cells (980 rpm, 10 min) and resuspend in pre-warmed MT. Add 65 ul of cells to each 96-well plate. Depending on the degranulation activity for each particular CHMC donor, load 1000-1500 cells/well. Mix four times followed by a 1 hr incubation at 37° C. During the 1 hr incubation, prepare 6× anti-IgE solution [rabbit anti-human IgE (1 mg/ml, Bethyl Laboratories A80-109A) diluted 1:167 in MT buffer]. Stimulate cells by adding 25 ul of 6× anti-IgE solution to the appropriate plates. Add 25 ul MT to un-stimulated control wells. Mix twice following addition of the anti-IgE. Incubate at 37° C. for 30 minutes. During the 30 minute incubation, dilute the 20 mM tryptase substrate stock solution [(Z-Ala-Lys-Arg-AMC 2TFA; Enzyme Systems Products, #AMC-246)] 1:2000 in tryptase assay buffer [0.1 M Hepes (pH 7.5), 10% w/v Glycerol, 10 uM Heparin (Sigma H-4898) 0.01% NaN₃]. Spin plates at 1000 rpm for 10 min to pellet cells. Transfer 25 ul of supernatant to a 96-well black bottom plate and add 100 ul of freshly diluted tryptase substrate solution to each well. Incubate plates at room temperature for 30 min. Read the optical density of the plates at 355 nm/460 nm on a spectrophotometric plate reader.

Leukotriene C4 (LTC4) is also quantified using an ELISA kit on appropriately diluted supernatant samples (determined empirically for each donor cell population so that the sample measurement falls within the standard curve) following the supplier's instructions.

7.5.3 CHMC High Cell Density IgE Activation Degranulation (Tryptase, Histamine), Leukotriene (LTC4), and Cytokine (TNFalpha, IL-13) Assays

Cultured human mast cells (CHMC) are sensitized for 5 days with IL-4 (20 ng/ml), SCF (200 ng/ml), IL-6 (200 ng/ml), and Human IgE (CP 1035K from Cortx Biochem, 100-500 ng/ml depending on generation) in CM medium. After sensitizing, cells are counted, pelleted (1000 rpm, 5-10 minutes), and resuspended at 1-2×10⁶ cells/ml in MT buffer. Add 100 ul of cell suspension to each well and 100 ul of compound dilutions. The final vehicle concentration is 0.5% DMSO. Incubate at 37° C. (5% CO₂) for 1 hour. After 1 hour of compound treatment, stimulate cells with 6× anti-IgE. Mix wells with the cells and allow plates to incubate at 37° C. (5% CO₂) for one hour. After 1 hour incubation, pellet cells (10 minutes, 1000 RPM) and collect 200 ul per well of the supernatant, being careful not to disturb pellet. Place the supernatant plate on ice. During the 7-hour step (see next) perform tryptase assay on supernatant that had been diluted 1:500. Resuspend cell pellet in 240 ul of CM media containing 0.5% DMSO and corresponding concentration of compound. Incubate CHMC cells for 7 hours at 37° C. (5% CO₂). After incubation, pellet cells (1000 RPM, 10 minutes) and collect 225 ul per well and place in -80° C. until ready to perform ELISAS. ELISAS are performed on appropriately diluted samples (determined empirically for each donor cell population so that the sample measurement falls within the standard curve) following the supplier's instructions.

7.5.4 BMMC High Cell Density IgE Activation Degranulation (Hexosimimidase, Histamine), Leukotriene (LTC4), and Cytokine (TNFalpha, IL-6) Assays 7.5.4.1 Preparation of WEHI-Conditioned Medium

WEHI-conditioned medium was obtained by growing murine myelomonocytic WEHI-3B cells (American Type Culture Collection, Rockville, Md.) in Iscove's Modified Eagles Media (Mediatech, Hernandon, Va.) supplemented with 10% heat-inactivated fetal bovine serum (FBS; JRH Biosciences, Kansas City, Mo.), 50 μM 2-mercaptoethanol (Sigma, St. Louis, Mo.) and 100 IU/mL penicillin-streptomycin (Mediatech) in a humidified 37° C., 5% CO₂/95% air incubator. An initial cell suspension was seeded at 200,000 cells/mL and then split 1:4 every 3-4 days over a period of two weeks. Cell-free supernatants were harvested, aliquoted and stored at −80° C. until needed.

7.5.4.2 Preparation of BMMC Medium

BMMC media consists of 20% WEHI-conditioned media, 10% heat-inactivated FBS (JHR Biosciences), 25 mM HEPES, pH7.4 (Sigma), 2 mM L-glutamine (Mediatech), 0.1 mM non-essential amino acids (Mediatech), 1 mM sodium pyruvate (Mediatech), 50 μM 2-mercaptoethanol (Sigma) and 100 IU/mL penicillin-streptomycin (Mediatech) in RPMI 1640 media (Mediatech). To prepare the BMMC Media, all components are added to a sterile IL filter unit and filtered through a 0.2 μm filter prior to use.

7.5.4.3 Protocol

Bone marrow derived mast cells (BMMC) are sensitized overnight with murine SCF (20 ng/ml) and monoclonal anti-DNP (10 ng/ml, Clone SPE-7, Sigma # D-8406) in BMMC media at a cell density of 666×10³ cells/ml. After sensitizing, cells are counted, pelleted (1000 rpm, 5-10 minutes), and resuspended at 1-3×10⁶ cells/ml in MT buffer. Add 100 ul of cell suspension to each well and 100 ul of compound dilutions. The final vehicle concentration is 0.5% DMSO. Incubate at 37° C. (5% CO₂) for 1 hour. After 1 hour of compound treatment, stimulate cells with 6× stimulus (60 ng/ml DNP-BSA). Mix wells with the cells and allow plates to incubate at 37° C. (5% CO₂) for one hour. After 1 hour incubation, pellet cells (10 minutes, 1000 RPM) and collect 200 ul per well of the supernatant, being careful not to disturb pellet, and transfer to a clean tube or 96-well plate. Place the supernatant plate on ice. During the 4-5 hour step (see next) perform the hexosimimidase assay. Resuspend cell pellet in 240 ul WEI-conditioned media containing 0.5% DMSO and corresponding concentration of compound. Incubate BMMC cells for 4-5 hours at 37° C. (5% CO₂). After incubation, pellet cells (1000 RPM, 10 minutes) and collect 225 ul per well and place in -80° C. until ready to perform ELISAS. ELISAS are performed on appropriately diluted samples (determined empirically for each donor cell population so that the sample measurement falls within the standard curve) following the supplier's instructions.

Hexosaminidase Assay:

In a solid black 96-well assay plate, add 50 uL hexosaminidase substrate (4-methylumbelliferyl-N-acetyl-13-D-glucosaminide; 2 mM) to each well. Add 50 uL of BMMC cell supernatant (see above) to the hexoseaminidase substrate, place at 37° C. for 30 minutes and read the plate at 5, 10, 15, and 30 minutes on a spectrophotometer.

7.5.5 Basophil IgE or Dustmite Activation Histamine Release Assay

The basophil activation assay was carried out using whole human peripheral blood from donors allergic to dust mites with the majority of the red blood cells removed by dextran sedimentation. Human peripheral blood was mixed 1:1 with 3% dextran T500 and RBCs were allowed to settle for 20-25 min. The upper fraction was diluted with 3 volumes of D-PBS and cells were spun down for 10 min at 1500 rpm, RT. Supernatant was aspirated and cells were washed in an equal volume MT-buffer. Finally, cells were resuspended in MT-buffer containing 0.5% DMSO in the original blood volume. 80 uL cells were mixed with 20 uL compound in the presence of 0.5% DMSO, in triplicate, in a V-bottom 96-well tissue culture plate. A dose range of 8 compound concentrations was tested resulting in a 10-point dose response curve including maximum (stimulated) and minimum (unstimulated) response. Cells were incubated with compound for 1 hour at 37° C., 5% CO₂ after which 20 uL of 6× stimulus [1 ug/mL anti-IgE (Bethyl Laboratories) 667 au/mL house dustmite (Antigen Laboratories)] was added. The cells were stimulated for 30 minutes at 37° C., 5% CO₂. The plate was spun for 10 min at 1500 rpm at room temperature and 80 uL the supernatant was harvested for histamine content analysis using the histamine ELISA kit supplied by Immunotech. The ELISA was performed according to supplier's instructions.

7.5.6 Results

The results of low density CHMC assays (Section 7.5.2), the high density BMMC assays (Section 7.5.4) and the basophil assays (Section 7.5.5) are provided in TABLE 1. The results of the high density CHMC assays (Section 7.5.3) are provided in TABLE 2. In TABLES 1 and 2, all reported values are IC₅₀s (in μM). A value of “9999” indicates an IC₅₀>10 μM, with no measurable activity at a 10 μM concentration. Most compounds tested had IC₅₀s of less than 10 μM, with many exhibiting IC₅₀s in the sub-micromolar range.

7.6 The 2,4-Pyrimidinediamine Compounds Inhibit FcγRI Receptor-Mediated Degranulation

The ability of the 2,4-pyrimidinediamine compounds of the invention to inhibit FcγRI-mediated degranulation was demonstrated with Compounds R921218, R921302, R921303, R940347, R920410, R927050, R940350, R935372, R920323, R926971 and R940352 in assays similar to those described in Section 7.5, with the exception that the cells were not primed with IgE and were activated with rabbit anti-human IgG Fab fragment (Bethyl Laboratories, Catalog No. A80-105).

All of the compounds tested exhibited IC₅₀s in the sub micromolar range.

TABLE 1 Low Density CHMC CHMC CHMC CHMC CHMC Basophils Basophils Basophils Test anti-IgE Ionomycin anti-IgE anti-IgE Ionomycin anti-IgE Ionomycin Dust mite Compound Tryptase Tryptase LTC4 Hexos. Hexos. Histamine Histamine Histamine R008951 R008952 R008953 R008955 R008956 R008958 R067934 R067963 R070153 R070790 1.665 9999 R070791 R081166 R088814 R088815 R091880 R092788 R908696 3.553 R908697 9999 9999 R909236 0.996 9999 R909237 9999 9999 R909238 0.174 9999 R909239 0.264 9999 R909240 0.262 9999 R909241 0.181 9999 R909242 0.567 9999 R909243 0.263 >10 R909245 0.255 6.242 R909246 0.169 9999 R909247 2.393 9999 R909248 3.582 9999 R909249 9999 9999 R909250 8.025 9999 R909251 0.138 9999 R909252 0.248 9999 R909253 7.955 9999 R909254 0.136 9999 R920664 9999 9999 R920665 1.1 9999 R920666 2.53 9999 R920668 3.2 9999 R920669 0.42 9999 R920670 2.18 9999 R920671 9999 9999 R920672 9999 9999 R920818 9999 9999 R920819 10 9999 R920820 9999 9999 R920846 9999 9999 R920860 1.009 9999 R920861 0.598 >10 R920893 1.239 9999 R920894 0.888 5.566 R920910 0.751 7.922 R920917 1.579 9.729 R921218 0.499 9999 0.55 0.6 9999 0.24 9999 0.302 R921219 0.059 9999 0.025 9999 0.020 R925734 9.2 >10 R925747 1.021 3.1 R925755 0.898 9999 R925757 2.8 9999 R925758 1.175 9999 R925760 4.85 9999 R925765 6.8 9999 R925766 8.9 9999 R925767 10 R925768 9999 R925769 9999 R925770 9999 R925771 0.5 2.8 0.22 R925772 9999 9999 R925773 0.673 9999 R925774 0.435 9999 R925775 0.225 9999 0.2 R925776 2.1 9999 R925778 0.225 9999 0.18 R925779 0.265 9999 0.19 R925783 2.9 9999 R925784 3.2 9999 R925785 2.5 9999 R925786 1.85 9999 R925787 9 9999 R925788 2.4 9999 R925790 9999 9999 R925791 9999 9999 R925792 6.25 9999 R925794 9999 9999 R925795 9999 9999 R925796 2 9999 R925797 0.85 9999 0.28 R925798 9999 9999 R925799 9999 9999 R925800 9999 9999 R925801 9999 9999 R925802 9999 9999 R925803 9999 9999 R925804 9999 9999 R925805 9999 9999 R925806 9999 9999 R925807 9999 9999 R925808 9999 9999 R925810 9999 9999 R925811 3.3 9999 R925812 5.8 9999 R925813 9999 9999 R925814 9999 9999 R925815 9999 9999 R925816 6 9999 R925819 9999 9999 R925820 9999 9999 R925821 9999 9999 R925822 9999 9999 R925823 9999 9999 R925824 9999 9999 R925837 9999 9999 R925838 9999 9999 R925839 9999 9999 R925840 9999 9999 R925841 9999 9999 R925842 7.3 9999 R925843 9999 9999 R925844 5.1 9999 R925845 2.3 9999 R925846 9999 9999 R925849 8.2 9999 R925851 0.925 9999 R925852 3 9999 R925853 9999 9999 R925854 9999 9999 R925855 4.2 9999 R925856 9.85 9999 R925857 5.95 9999 R925858 8.05 7.3 R925859 9999 9999 R925860 9999 9999 R925861 9999 9999 R925862 0.7 9999 R925863 0.274 9999 R925864 9999 9999 R925865 9999 9999 R926016 9999 9999 R926017 1.43 9999 0.53 9999 R926018 9999 10 R926037 9999 9999 R926038 9999 9999 R926039 9999 9999 R926058 9999 9999 R926064 6.2 R926065 3.5 R926068 >10 R926069 9.1 R926072 >10 R926086 2.5 9999 R926108 0.76 0.787 6.4 0.95 9999 R926109 0.538 5.5 0.73 0.55 >10 0.15 9999 R926110 1.071 9999 1.42 1.2 >10 0.3 9999 R926113 0.413 0.49 0.413 9999 0.27 9999 R926114 3.427 8.1 1.7 10 R926145 4.764 >10 R926146 1.59 0.761 6.7 R926147 1.899 >10 R926206 >10 >10 R926209 >10 9999 R926210 0.926 9999 0.8 700 9999 0.37 >10 R926211 1.299 9.8 2.7 9999 1.55 >10 R926212 0.654 9999 0.45 0.5 >10 R926213 1.639 5.5 1.75 >10 R926218 >10 R926219 1.102 6.7 R926220 >10 R926221 8.5 R926222 >10 R926223 >10 R926224 >10 R926225 >10 R926228 >10 R926229 >10 R926230 >10 R926234 >10 R926237 1.207 6.2 R926240 0.381 1.7 0.145 R926241 7 9999 R926242 4.2 9999 R926243 3.1 9999 R926245 3.1 9.4 R926248 0.9 9999 0.76 R926249 0.5 9999 0.25 R926252 2.8 R926253 0.8 0.675 R926254 1.3 4 R926255 1.4 4.5 R926256 0.275 5.1 0.23 R926257 1.5 7.5 R926258 0.9 9999 0.59 R926259 2.5 6.2 R926319 9999 9999 R926320 9999 9999 R926321 9999 9999 R926325 9999 9999 R926331 9999 9999 R926339 0.66 9999 R926340 3.23 9999 R926341 0.875 9999 R926342 10 9999 R926376 9999 R926386 9999 9999 R926387 0.65 9999 0.7 R926394 9999 9999 R926395 0.875 6.4 0.29 R926396 0.7 2.6 0.16 R926397 9999 9999 R926398 9999 9999 R926399 9999 9999 R926400 9999 9999 R926401 9999 9999 R926402 9999 9999 R926403 9999 9999 R926404 9999 9999 R926405 3.4 9999 R926406 9999 9999 R926408 9.6 9999 R926409 3.15 9999 R926411 0.69 2.5 R926412 0.62 9999 R926461 0.725 9999 R926467 1.175 8.8 R926469 9999 R926474 2.5 9999 R926475 2.15 >10 R926476 0.6 7.7 R926477 0.27 9999 R926478 9999 R926479 9999 R926480 1.9 9999 R926481 1.445 9999 R926482 1.037 >10 R926483 9999 R926484 1.523 9999 R926485 4.012 9999 R926486 0.647 7.403 R926487 0.554 8.867 1.25 R926488 0.331 >10 0.752 R926489 1.414 >10 R926490 1.571 9999 R926491 1.158 >10 R926492 0.645 9999 R926493 0.25 9.181 0.078 R926494 0.313 9999 0.078 R926495 0.121 >10 0.078 0.04 9999 0.038 R926496 0.571 >10 R926497 0.138 9999 0.27 9999 0.205 R926498 0.209 >10 R926499 0.29 >10 R926500 0.418 >10 R926501 0.298 >10 0.609 9999 0.645 R926502 0.483 >10 0.405 9999 0.491 R926503 0.452 >10 R926504 0.569 >10 R926505 0.145 9999 R926506 0.343 9999 R926508 0.127 9999 0.065 9999 0.054 R926509 1.16 9999 R926510 0.44 >10 R926511 0.786 >10 R926514 9999 9999 R926516 1 9999 R926526 9999 9999 R926527 9999 9999 R926528 8.75 9999 R926535 9999 9999 R926536 9999 9999 R926555 9999 9999 R926559 7.7 9999 R926560 9999 9999 R926562 9999 9999 R926563 9999 9999 R926564 3.75 9999 R926565 0.625 3.3 R926566 2.73 9999 R926567 9.3 9999 R926569 0.61 3.07 R926571 9999 9999 R926572 1.8 6.08 R926574 1.96 2.63 R926576 9999 9999 R926579 9999 9999 R926580 10 9999 R926582 1.3 9999 R926583 9999 9999 R926584 9999 9999 R926585 9999 9999 R926586 2.75 9999 R926587 9999 9999 R926588 7.85 9999 R926589 0.325 10 R926591 2.62 9999 R926593 0.68 8.3 0.495 R926594 9999 9999 R926595 4.85 9999 R926604 2.85 9999 R926605 2.45 9999 R926614 0.228 9999 R926615 0.445 9999 R926616 0.625 3.25 R926617 9.45 9999 R926620 8.35 9999 R926623 9999 9999 R926662 9999 9999 R926663 9999 9999 R926675 0.63 9999 R926676 0.76 9999 R926680 1.71 9999 R926681 0.775 9999 R926682 8.41 9999 R926683 10 9999 R926688 2.25 >10 R926690 0.146 >10 R926696 0.309 >10 R926698 9999 R926699 0.76 9999 R926700 0.157 >10 R926701 2.2 9999 R926702 0.886 9999 R926703 0.525 9999 R926704 0.564 9999 R926705 0.263 9999 0.533 R926706 0.07 2.406 0.078 R926707 0.214 9999 R926708 0.472 9999 R926709 0.858 9999 R926710 1.763 9999 R926711 1.245 9999 R926712 1.084 9999 R926713 0.446 8.741 R926714 0.428 >10 R926715 0.588 >10 R926716 1.06 9999 R926717 7.874 9999 R926718 1.826 9999 R926719 0.1335 4.024 R926720 1.555 9999 R926721 4.441 9999 R926722 5.96 9999 R926723 2.591 9999 R926724 2.059 9999 R926725 0.431 9999 R926726 9999 9999 R926727 0.387 9999 R926728 0.482 >10 R926730 0.251 9999 R926731 9999 9999 R926732 0.444 9999 R926733 1.496 9999 R926734 4.493 9999 R926735 3.712 9999 R926736 0.288 9999 R926737 0.059 9999 R926738 0.342 9999 R926739 0.508 9999 R926740 4.422 9999 R926741 2.908 9999 R926742 0.127 0.043 9999 0.055 R926743 9999 R926744 9999 R926745 0.083 9999 R926746 0.989 9999 R926747 0.213 >10 R926748 0.345 >10 R926749 0.472 9999 R926750 0.361 >10 R926751 0.598 9999 R926764 0.252 5.64 R926765 0.324 4.39 R926766 0.756 9999 R926767 0.387 >10 R926768 0.443 >10 R926769 1.067 9999 R926770 0.583 9999 R926771 2.049 9999 R926772 0.337 7.501 R926773 0.548 7.849 R926774 1.934 7.935 R926775 3.47 >10 R926776 0.81 9999 R926777 0.378 9999 R926778 0.414 9999 R926779 9999 9999 R926780 0.152 >10 R926781 0.573 9999 R926782 0.173 >10 R926783 0.304 >10 R926784 0.252 9999 R926785 0.222 >10 R926786 0.504 9999 R926787 5.422 9999 R926788 0.336 6.341 R926789 2.315 9999 R926790 0.462 7.412 R926791 0.233 >10 R926792 3.197 9999 R926793 3.073 9999 R926795 2.041 >10 R926796 0.914 9999 R926797 2.235 9999 R926798 2.347 5.87 R926799 9999 9999 R926800 4.581 9999 R926801 10 9999 R926802 1.251 >10 R926803 1.541 >10 R926804 1.578 7.109 R926805 0.764 9999 R926806 0.374 9999 R926807 0.291 9999 R926808 0.368 9999 R926809 0.78 3.052 R926810 1.221 9999 R926811 3.662 9999 R926812 0.185 >10 R926813 0.152 9999 R926814 1.101 9999 R926815 1.181 9999 R926816 0.084 9999 R935000 9999 9999 R935001 9999 9999 R935002 9999 9999 R935003 9999 9999 R935004 9999 9999 R935005 9999 9999 R935006 10 9.8 R935016 9999 9999 R935019 8.8 9999 R935020 9999 9999 R935021 9999 9999 R935023 9999 9999 R935025 1.04 9999 R935029 2.83 9999 R935075 0.93 9999 R935076 4.15 9999 R935077 9999 9999 R935114 1.725 9999 R935117 9999 R935134 0.909 1.799 R935135 10 9999 R935136 0.952 2.129 R935137 10 9999 R935138 0.096 0.552 R935139 0.846 9999 R935140 0.275 0.959 R935141 0.727 >10 R935142 0.873 >10 R935143 0.573 >10 R935144 0.63 9999 R935145 0.548 >10 R935146 3.802 9999 R935147 1.404 9999 R935148 2.218 9.423 R935149 0.708 >10 R935150 1.926 9.738 R935151 0.479 >10 R935152 0.505 9.316 R935153 0.238 >10 R935154 0.127 >10 R935155 0.401 9999 R935156 0.149 >10 R935157 0.256 4.656 R935158 0.551 >10 R935159 0.232 4.135 R935160 0.202 >10 R935161 0.277 9999 R935162 0.269 >10 R935163 9999 9999 R935164 0.204 9999 R935165 4.988 9999 R935166 0.568 9999 R935167 2.132 >10 R935168 0.488 9.484 R935169 0.999 8.007 R935170 0.673 9999 R935171 0.536 9999 R935172 1.385 6.808 R935173 0.454 >10 R935174 1.384 9999 R935175 0.885 9999 R935176 1.169 9999 R935177 0.889 >10 R935178 0.515 9999 R935179 0.557 9999 R935180 1.22 9999 R935181 1.76 9999 R935182 0.124 2.469 R935183 0.729 9999 R935184 0.605 9999 R935185 0.351 6.642 R935186 0.211 9999 R935187 9.059 >10 R935188 0.239 9999 R935189 0.619 9999 R935190 0.156 9999 R935191 0.151 9999 R935192 0.337 9999 R935193 0.136 9999 R935194 0.11 9999 R935196 0.117 9999 R935197 0.174 >10 R935198 0.126 >10 R935199 0.45 >10 R935202 0.181 9.765 R935203 0.562 >10 R935204 0.554 9999 R935205 2.959 9999 R935206 4.711 9999 R935207 9999 9999 R935208 1.274 9999 R935209 0.526 1.035 R935211 1.238 9999 R935212 1.427 9999 R935213 0.619 10 R935214 0.453 5.499 R935218 4.712 9999 R935219 5.409 9999 R935220 3.789 9999 R940089 9999 9999 R940090 9999 9999 R940095 9999 9999 R940100 9999 9999 R940215 0.845 9999 R940216 0.2675 7.3 R940217 9999 9999 R940222 9999 9999 R940233 0.132 >10 R940235 0.8 >10 R940250 R940251 R940253 1.006 >10 R940254 0.986 9999 R940255 1.033 9999 R940256 1.104 9999 R940257 0.667 9999 R940258 0.473 5.72 R940260 1.126 9999 R940261 9999 9999 R940262 9999 9999 R940263 9999 9999 R940264 10 9999 R940265 0.239 >10 R940266 9999 9999 R940267 3.151 9999 R940269 1.654 9999 R940270 2.144 8.739 R940271 0.401 6.821 R940275 0.862 9999 R940276 0.211 9999 R940277 0.141 9999 R940280 6.999 9999 R940281 0.525 5.529 R940282 0.401 3.015 R940283 0.553 4.982 R940284 0.465 3.744 R940285 3.499 9999 R940286 0.337 7.082 R940287 0.288 7.684 R940288 0.208 9999 R940289 0.272 9999 R940290 0.116 9999 R940291 0.396 9999 R940292 0.683 9999 R940293 9999 9999 R940294 1.366 9999 R940295 0.126 8.812 R940296 0.41 >10 R940297 3.465 10 R945025 9999 9999 R945032 0.37 9999 R945033 9999 9999 R945034 1.85 9999 R945035 9999 9999 R945036 9999 9999 R945037 9999 9999 R945038 9999 9999 R945040 9999 9999 R945041 9999 9999 R945042 9999 9999 R945043 9999 9999 R945045 9999 9999 R945046 0.82 >10 R945047 0.845 9999 R945048 0.76 9999 R945051 0.95 >10 R945052 0.425 2.48 R945053 0.1185 1.48 R945056 10 9999 R945057 10 9999 R945060 0.9375 >10 R945061 10 9999 R945062 0.625 >10 R945063 1.55 >10 R945064 0.53 >10 R945065 1.425 >10 R945066 5.2 nd R945067 9999 nd R945068 9999 nd R945070 0.45 >10 R945071 0.205 >10 R945096 1.75 >10 R945097 10 9999 R945109 1.025 >10 R945110 0.602 9999 R945117 4.077 9999 R945118 0.668 9999 R945124 0.69 7.852 R945125 0.896 >10 R945126 9999 9999 R945127 0.704 8.955 R945128 0.685 8.8 R945129 1.003 >10 R945130 1.874 9999 R945131 0.77 9999 R945132 0.571 8.77 R945133 1.064 >10 R945134 9999 9999 R945135 0.986 8.245 R945137 1.649 >10 R945138 1.058 6.733 R945139 1.016 >10 R945140 0.573 >10 R945142 1.049 >10 R945144 0.244 9999 R945145 9999 >10 R945146 3.756 9999 R945147 3.546 9999 R945148 0.307 9999 R945149 0.391 >10 R945150 0.467 >10 R945151 4.07 9999 R945152 6.94 9999 R945153 0.688 6.561 R945155 1.878 >10 R945156 0.787 9999 R945157 1.477 9999 R945162 9999 9999 R945163 0.922 4.251 R945164 10 9999 R945165 9999 9999 R945166 9999 9999 R945167 0.761 9999 R945168 10 9999 R945169 10 9999 R945170 0.661 >10 R945171 1.327 9999 R945172 1.179 9999 R945173 1.419 9999 R945175 1.648 9999 R950082 9999 9999 R950083 9999 9999 R950090 9999 9999 R921302 0.37 9999 0.19 9999 0.282 R950092 9999 9999 R950093 0.64 5.55 R950100 0.71 >10 R950107 0.46 >10 R950108 2.075 >10 R950109 7.95 R950120 3 9999 R950121 4.25 >10 R950122 3.025 9999 R950123 3.25 8.45 R950125 1.375 6.3 R950129 0.665 >10 R950130 4.9 R950131 9999 R950132 9 R950133 2.2 >10 R950134 1.875 9999 R950135 0.85 >10 R950137 2.23 9999 R950138 9.5 R950139 1.375 9999 R950140 2.825 9999 R950141 0.31 >10 R950142 10 R950143 8.23 R950144 10 R950145 9999 R950146 9999 R950147 9999 R950148 2.275 9999 R950149 10 9999 R950150 9999 9999 R950151 9999 R950152 10 R950153 9999 R950154 2.075 9999 R950155 9999 R950156 9999 R950157 9999 R950158 9.98 R950159 0.61 9999 R950160 1 9999 R950162 0.434 >10 R950163 0.874 9999 R950164 1.893 9999 R950165 1.288 9999 R950166 1.889 9999 R950167 9999 9999 R950168 6.496 8.653 R950169 1.273 9.518 R950170 9999 9999 R950171 0.585 >10 R950172 0.983 9999 R950173 2.368 >10 R950174 4.618 9999 R950175 1.688 9999 R950176 1.342 9999 R950177 2.361 8.434 R950178 0.688 >10 R950179 0.955 >10 R950180 0.278 9999 R950181 0.254 9999 R950182 0.627 9999 R950183 4.797 9999 R950184 2.222 9999 R950185 1.03 8.81 R950186 0.558 >10 R950187 0.724 >10 R950188 2.327 9999 R950189 10 9999 R950190 1.573 9999 R950191 0.178 9999 R950192 0.244 9999 R950193 0.61 9999 R950194 2.04 9999 R950195 0.473 9999 R950196 2.2 9999 R950197 0.531 9999 R950198 0.406 >10 R950199 0.408 9999 R950200 0.245 9999 R950201 0.261 9999 R950202 3.218 9999 R950203 9.035 9999 R950204 6.285 9999 R950205 8.997 9999 R950206 3.66 >10 R950207 0.164 9999 R950208 0.267 9999 R950209 0.748 9999 R950210 10 9999 R950211 10 9999 R950212 0.253 9999 R950213 9999 9999 R950214 10 9999 R950215 0.409 9999 R950216 0.327 9999 R950217 0.34 9999 R950218 0.292 9999 R950219 0.439 9999 R950220 0.489 9999 R950221 0.636 9999 R950222 0.865 9999 R950223 0.763 9999 R950224 0.687 9999 R950225 5.283 9999 R950226 1.374 9999 R950227 1.029 9999 R950229 0.98 9999 R950230 7.91 9999 R950231 1.968 9999 R950232 10 9999 R950233 0.98 9999 R950234 10 9999 R950235 4.095 9999 R950236 0.955 9999 R950237 9999 9999 R950238 10 9999 R950239 2.063 9999 R950240 1.766 9999 R950241 3.275 9999 R950251 9999 9999 R950253 0.697 9999 R950254 0.496 9999 R950255 10 9999 R908698 1.67 9999 R908699 0.217 9999 R908700 1.273 9999 R908701 0.099 7.643 R908702 0.104 7.395 R908703 0.63 9999 R908704 0.511 9999 R908705 0.801 9999 R908706 0.445 9999 R908707 1.834 9999 R908709 2.414 R908710 1.838 99 R908711 1.761 R908712 0.075 99 R908734 1.379 R909255 0.244 9999 R909259 0.43 9999 R909260 1.041 9999 R909261 0.93 9999 R909263 0.289 9999 R909264 R909265 99 R909266 99 R909267 0.589 9999 R909268 0.071 9999 R909290 0.226 R909292 1.172 R909308 0.671 9999 R909309 0.083 9999 R920394 R920395 0.092 9999 R920396 R920397 R920398 R920399 R920404 R920405 R920406 R920407 R920408 R920410 0.125 9999 R920411 0.564 9999 R925745 1.766 9999 R926238 9999 R926752 0.338 9999 R926753 0.108 9999 R926754 0.388 9999 R926755 1.693 9999 R926756 1.365 9999 R926757 0.158 9999 R926759 0.688 9999 R926760 2.893 9999 R926761 0.245 9999 R926762 0.386 9999 R926763 0.195 9999 R926794 1.382 9999 R926826 0.613 9999 R926827 1.098 9999 R926828 0.306 9999 R926829 0.688 9999 R926830 0.569 10 R926831 0.133 10 R926832 0.365 9999 R926833 1.129 9999 R926834 0.145 9999 R926835 0.296 9999 R926836 10 9999 R926837 2.994 9999 R926838 0.583 9999 R926839 0.161 9999 R926840 1.1 9999 R926841 0.551 9999 R926842 7.733 9999 R926843 7.371 9999 R926844 1.1 9999 R926845 2.558 7.812 R926846 0.86 6.264 R926847 1.479 6.264 R926848 0.254 10 R926851 0.446 R926855 9999 9999 R926856 0.734 9999 R926857 1.209 9999 R926859 R926860 1.949 99 R926862 0.774 9999 R926863 R926866 R926870 3.294 R926871 2.146 R926874 0.638 9999 R926879 0.397 9999 R926880 R926881 R926883 R926885 R926886 R926887 1.747 R926890 0.361 9999 R926891 0.152 9999 R926892 0.685 9999 R926893 10 9999 R926894 9999 9999 R926895 0.339 9999 R926896 1.622 9999 R926897 1.727 9999 R926898 1.1 9999 R926899 1.1 9999 R926900 9999 9999 R926902 1.37 4.586 R926903 0.243 9999 R926904 0.538 R926905 99 R926906 0.794 R926907 0.764 R926908 0.585 R926909 0.379 R926913 0.548 9999 R926914 1.86 9999 R926915 1.713 9999 R926916 1.958 9999 R926917 1.169 9999 R926918 2.521 9999 R926919 1.413 9999 R926922 0.305 9999 R926923 0.346 9999 R926925 0.307 99 R926926 0.401 9999 R926927 0.348 9999 R926928 0.575 9999 R926929 1.916 9999 R926930 99 9999 R926931 R926932 0.31 9999 R926933 R926934 R926935 4.44 R926936 R926937 R926938 R926939 3.615 R926940 7.754 R926941 4.195 R926942 4.81 R926943 R926944 0.225 99 R926945 0.457 9999 R926946 R926947 0.354 9999 R926948 0.246 9999 R926949 0.089 9999 R926950 99 9999 R926951 0.183 9999 R926953 0.049 9999 R926954 0.284 9999 R926955 0.36 9999 R926956 0.211 9999 R927016 1.408 R927017 2.449 R927018 1.446 R927019 1.179 R927020 1.316 9999 R927023 0.918 9999 R935221 9999 9999 R935222 0.52 9999 R935223 0.469 9999 R935224 4.578 9999 R935225 6.495 9999 R935237 0.24 9999 R935238 1.854 9999 R935239 0.609 9999 R935240 0.606 9999 R935242 2.855 9999 R935248 1.1 9999 R935249 1.1 9999 R935250 1.1 9999 R935251 R935252 R935253 R935255 0.374 9999 R935256 0.324 9999 R935258 1.191 9999 R935259 1.777 9999 R935261 0.391 9999 R935262 0.516 9999 R935263 0.106 10 R935264 0.135 9999 R935266 2.97 R935267 2.463 R935268 1.059 R935269 1.715 R935271 R935276 2.33 R935277 22.883 8.9 R935278 4.753 9999 R935279 0.889 9999 R935280 99 R935281 1.399 9999 R935286 1.158 9999 R935287 0.403 9999 R935288 1.58 9999 R935289 1.688 9999 R935290 0.34 9999 R935291 1.364 9999 R935292 0.483 9999 R935293 0.141 9999 R935294 0.388 9999 R935295 1.943 9999 R935296 99 9999 R935297 7.328 9999 R935298 0.252 99 R935299 0.21 9999 R935300 0.243 9999 R935301 4.05 99 R935302 0.189 9999 R935303 0.244 99 R935304 0.188 9999 R935305 0.495 9999 R935306 0.345 99 R935307 0.139 99 R935308 0.275 9999 R935309 R935310 R935320 2.769 R935321 2.986 R935322 3.416 R935323 9999 R935324 9999 R935336 0.341 9999 R935337 9999 R935338 0.411 9999 R935339 9999 R935340 3.606 R935351 9999 9999 R935352 R935353 9999 9999 R935354 99 9999 R935355 9999 9999 R935356 99 R935357 99 9999 R935358 9999 9999 R935359 1.027 9999 R935360 0.903 9999 R935361 1.438 9999 R935362 0.409 9999 R935363 0.405 9999 R935364 0.563 9999 R935365 0.373 9999 R935366 0.216 9999 R935367 0.053 9999 R940079 9999 R940110 9999 9999 R940299 2.497 9999 R940300 10 9999 R940301 1.975 9999 R940304 9999 9999 R940306 1.1 9999 R940307 0.291 9999 R940308 0.612 4.168 R940309 1.132 9999 R940311 1.95 R940312 2.557 R940314 4.197 R940316 1.858 R940317 0.913 9999 R940318 3.792 R940319 9999 R940321 9999 R940323 0.048 9999 R940337 1.098 R940338 0.073 9999 R921303 0.033 99 R940345 1.712 R940346 0.142 99 R940347 0.063 99 R940348 2.189 R940349 0.044 7.4 R940350 0.092 4 R940351 0.12 2.7 R940352 0.101 9999 R940353 0.091 9999 R940354 0.115 99 R945236 0.562 9999 R945237 0.461 9999 R945242 0.247 9999 R945263 1.642 R921304 0.085 9999 R945299 R950244 9999 R950245 9999 R950246 9999 R950247 9999 R950261 0.611 9999 R950262 0.285 9999 R950263 0.284 3.299 R950264 0.198 9999 R950265 0.312 9999 R950266 0.645 9999 R950267 0.18 9999 R950290 9999 9999 R950291 9999 9999 R950293 3.689 8.155 R950294 2.005 8.005 R950295 2.041 8.795 R950296 0.495 9999 R950344 99 R950345 1.962 99 R950346 0.345 9999 R950347 0.548 R950348 0.066 R950349 0.078 9999 R950356 R950368 0.038 9999 R950371 R950372 1.348 9999 R950373 R950374 0.599 9999 R950376 2.539 R950377 99 R950378 R950379 0.545 9999 R950380 3 9999 R950381 0.11 99 R950382 R950383 0.114 9999 R950385 R950386 0.973 R950388 2.518 R950389 0.612 9999 R950391 999 9999 R950392 0.956 9999 R950393 0.404 9999 R945028 R935241 R940298 R940302 R940303 R940305 R935260 9999 R909258 R940313 9999 R940315 9999 R935275 9999 R940320 9999 R940322 9999 9999 R926910 9999 9999 R926911 9999 9999 R926912 9999 9999 R926853 9999 9999 R926852 9999 9999 R926854 9999 9999 R926920 9999 9999 R926921 99 9999 R926924 99 9999 R926858 R926861 9999 9999 R945298 9999 9999 R940328 9999 R926869 R926873 9999 R926875 9999 R926876 9999 R926877 9999 R940336 9999 R926878 9999 R926882 9999 R926884 9999 R926889 9999 R920400 9999 R920401 9999 R920402 9999 R920403 9999 R940342 99 R920409 9999 R940344 9999 R926888 9999 R926758 R927024 0.326 99 R927025 0.326 R927026 9999 9999 R927027 9999 9999 R927028 0.208 9999 R927029 R927030 0.26 9999 R927031 0.215 99 R927032 0.899 R927035 0.583 9999 R927036 R927037 0.233 9999 R927038 1.05 9999 R927039 1.23 9999 R927040 1.05 9999 R927041 0.788 9999 R927042 R935270 R935368 0.082 9999 R935369 0.255 9999 R935370 R935371 0.794 9999 R935372 0.06 9999 R935373 0.274 9999 R935374 0.356 9999 R935375 10 9999 R935376 R935377 R935378 0.566 9999 R935379 R935380 1.61 99 High Density BMMC BMMC BMMC BMMC BMMC BMMC Test anti-IgE Ionomycin anti-IgE anti-IgE anti-IgE anti-IgE Compound hexos Hexos. histamine LTC4 TNF-alpha IL-6 R008951 R008952 R008953 R008955 R008956 R008958 R067934 R067963 R070153 R070790 R070791 R081166 R088814 R088815 R091880 R092788 R908696 R908697 R909236 R909237 R909238 <0.22 <0.22 0.521 0.432 <0.22 R909239 R909240 R909241 <0.22 <0.22 1.021 0.253 <0.22 R909242 R909243 R909245 R909246 R909247 R909248 R909249 R909250 R909251 R909252 R909253 R909254 R920664 R920665 R920666 R920668 R920669 R920670 R920671 R920672 R920818 R920819 R920820 R920846 R920860 R920861 R920893 R920894 R920910 R920917 R921218 0.133 9999 0.203 0.766 0.274 0.100 R921219 0.069 0.058 0.040 0.039 0.009 R925734 9999 9999 R925747 3.1 R925755 R925757 R925758 R925760 R925765 R925766 R925767 R925768 R925769 R925770 R925771 R925772 R925773 R925774 R925775 R925776 R925778 R925779 R925783 R925784 R925785 R925786 R925787 R925788 R925790 R925791 R925792 R925794 R925795 R925796 R925797 R925798 R925799 R925800 R925801 R925802 R925803 R925804 R925805 R925806 R925807 R925808 R925810 R925811 R925812 R925813 R925814 R925815 R925816 R925819 R925820 R925821 R925822 R925823 R925824 R925837 R925838 R925839 R925840 R925841 R925842 R925843 R925844 R925845 R925846 R925849 R925851 R925852 R925853 R925854 R925855 R925856 R925857 R925858 R925859 R925860 R925861 R925862 R925863 R925864 R925865 R926016 9999 9999 R926017 1.4 9.6 R926018 8.5 9999 R926037 9999 9999 R926038 9999 9999 R926039 9999 9999 R926058 9999 9999 R926064 5.9 7.3 R926065 9999 9999 R926068 7.4 8.2 R926069 4.5 4.4 R926072 9999 9999 R926086 2.8 7.3 R926108 0.9 9999 R926109 0.6 3.2 R926110 1 4.5 R926113 0.65 9999 R926114 9999 9999 R926145 2.4 8.8 R926146 1.35 5 R926147 2 7.1 R926206 6.6 8.6 R926209 10 9.1 R926210 0.6 >10 R926211 3.9 >10 R926212 0.5 5 R926213 R926218 9999 9999 R926219 2.5 3.2 R926220 9999 9999 R926221 9.9 9999 R926222 9999 9999 R926223 9999 9999 R926224 9999 9999 R926225 9999 9999 R926228 9999 R926229 R926230 R926234 9999 R926237 1.9 R926240 R926241 R926242 R926243 R926245 R926248 R926249 R926252 R926253 R926254 R926255 R926256 R926257 R926258 R926259 R926319 R926320 R926321 R926325 R926331 R926339 R926340 R926341 R926342 R926376 R926386 R926387 R926394 R926395 R926396 R926397 R926398 R926399 R926400 R926401 R926402 R926403 R926404 R926405 R926406 R926408 R926409 R926411 R926412 R926461 R926467 R926469 R926474 R926475 R926476 R926477 R926478 R926479 R926480 R926481 R926482 R926483 R926484 R926485 R926486 R926487 R926488 R926489 R926490 R926491 R926492 R926493 R926494 R926495 0.056 0.089 0.24 0.077 0.028 R926496 R926497 R926498 <0.22 0.515 0.995 0.614 <0.22 R926499 R926500 R926501 R926502 R926503 R926504 R926505 <0.22 <0.22 <0.22 <0.22 <0.22 R926506 R926508 0.086 0.107 0.162 0.054 0.026 R926509 R926510 R926511 R926514 R926516 R926526 R926527 R926528 R926535 R926536 R926555 R926559 R926560 R926562 R926563 R926564 R926565 R926566 R926567 R926569 R926571 R926572 R926574 R926576 R926579 R926580 R926582 R926583 R926584 R926585 R926586 R926587 R926588 R926589 R926591 R926593 R926594 R926595 R926604 R926605 R926614 R926615 R926616 R926617 R926620 R926623 R926662 R926663 R926675 R926676 R926680 R926681 R926682 R926683 R926688 R926690 R926696 R926698 R926699 R926700 R926701 R926702 R926703 R926704 R926705 R926706 R926707 <0.056 <0.056 0.39 0.088 <0.056 R926708 R926709 R926710 R926711 R926712 R926713 R926714 R926715 R926716 R926717 R926718 R926719 R926720 R926721 R926722 R926723 R926724 R926725 R926726 R926727 R926728 R926730 R926731 R926732 R926733 R926734 R926735 R926736 R926737 0.075 0.073 0.046 0.068 0.017 R926738 R926739 R926740 R926741 0.961 1.025 9999 0.772 0.537 R926742 0.041 0.055 0.105 0.053 0.022 R926743 R926744 R926745 R926746 R926747 R926748 R926749 R926750 R926751 R926764 R926765 R926766 R926767 R926768 R926769 R926770 R926771 R926772 R926773 R926774 R926775 R926776 R926777 R926778 R926779 R926780 <0.22 <0.22 0.461 <0.22 <0.22 R926781 R926782 <0.22 <0.22 1.461 0.276 <0.22 R926783 R926784 R926785 0.989 0.561 1.411 1.312 0.513 R926786 R926787 R926788 R926789 R926790 R926791 0.064 <0.056 0.896 0.205 <0.056 R926792 R926793 R926795 R926796 R926797 R926798 R926799 R926800 R926801 R926802 R926803 R926804 R926805 R926806 R926807 R926808 R926809 R926810 R926811 R926812 R926813 R926814 R926815 R926816 R935000 R935001 R935002 R935003 R935004 R935005 R935006 R935016 R935019 R935020 R935021 R935023 R935025 R935029 R935075 R935076 R935077 R935114 R935117 R935134 R935135 R935136 R935137 R935138 <0.22 <0.22 0.373 0.409 <0.22 R935139 R935140 R935141 R935142 R935143 R935144 R935145 R935146 R935147 R935148 R935149 R935150 R935151 R935152 R935153 R935154 0.104 0.085 0.547 0.131 0.041 R935155 R935156 <0.22 <0.22 0.433 0.22 <0.22 R935157 R935158 R935159 R935160 <0.22 0.317 0.876 0.484 <0.22 R935161 R935162 R935163 R935164 R935165 R935166 R935167 R935168 R935169 R935170 R935171 R935172 R935173 R935174 R935175 R935176 R935177 R935178 R935179 R935180 R935181 R935182 R935183 R935184 R935185 R935186 R935187 R935188 R935189 R935190 R935191 0.068 0.043 0.213 0.071 0.027 R935192 R935193 0.08 0.048 0.312 0.092 0.037 R935194 0.125 0.054 0.493 0.118 0.034 R935196 R935197 R935198 R935199 R935202 R935203 R935204 R935205 R935206 R935207 R935208 R935209 R935211 R935212 R935213 R935214 R935218 R935219 R935220 R940089 R940090 R940095 R940100 R940215 R940216 R940217 R940222 R940233 R940235 R940250 R940251 R940253 R940254 R940255 R940256 R940257 R940258 R940260 R940261 R940262 R940263 R940264 R940265 0.981 0.306 1.211 1.131 0.486 R940266 R940267 R940269 R940270 R940271 R940275 R940276 0.136 0.073 0.332 0.251 <0.056 R940277 0.279 0.315 0.625 0.262 0.181 R940280 R940281 R940282 R940283 R940284 R940285 R940286 R940287 R940288 R940289 R940290 0.255 0.545 0.59 0.246 0.1 R940291 R940292 R940293 R940294 R940295 R940296 R940297 R945025 R945032 R945033 R945034 R945035 R945036 R945037 R945038 R945040 R945041 R945042 R945043 R945045 R945046 R945047 R945048 R945051 R945052 R945053 R945056 R945057 R945060 R945061 R945062 R945063 R945064 R945065 R945066 R945067 R945068 R945070 R945071 R945096 R945097 R945109 R945110 R945117 R945118 R945124 R945125 R945126 R945127 R945128 R945129 R945130 R945131 R945132 R945133 R945134 R945135 R945137 R945138 R945139 R945140 R945142 R945144 R945145 R945146 R945147 R945148 R945149 R945150 >2 >2 9999 0.709 0.634 R945151 R945152 R945153 R945155 R945156 R945157 R945162 R945163 R945164 R945165 R945166 R945167 R945168 R945169 R945170 R945171 R945172 R945173 R945175 R950082 R950083 R950090 R921302 R950092 R950093 R950100 R950107 R950108 R950109 R950120 R950121 R950122 R950123 R950125 R950129 R950130 R950131 R950132 R950133 R950134 R950135 R950137 R950138 R950139 R950140 R950141 R950142 R950143 R950144 R950145 R950146 R950147 R950148 R950149 R950150 R950151 R950152 R950153 R950154 R950155 R950156 R950157 R950158 R950159 R950160 R950162 R950163 R950164 R950165 R950166 R950167 R950168 R950169 R950170 R950171 R950172 R950173 R950174 R950175 R950176 R950177 R950178 R950179 R950180 R950181 R950182 R950183 R950184 R950185 R950186 R950187 R950188 R950189 R950190 R950191 <0.22 >2 0.401 <0.22 <0.22 R950192 R950193 R950194 R950195 R950196 R950197 R950198 R950199 R950200 R950201 R950202 R950203 R950204 R950205 R950206 R950207 <0.22 <0.22 0.288 <0.22 <0.22 R950208 R950209 R950210 R950211 R950212 R950213 R950214 R950215 R950216 R950217 R950218 R950219 R950220 R950221 R950222 R950223 R950224 R950225 R950226 R950227 R950229 R950230 R950231 R950232 R950233 R950234 R950235 R950236 R950237 R950238 R950239 R950240 R950241 R950251 R950253 R950254 R950255 R908698 R908699 R908700 R908701 R908702 R908703 R908704 R908705 R908706 R908707 R908709 R908710 R908711 R908712 R908734 R909255 R909259 R909260 R909261 R909263 R909264 R909265 R909266 R909267 R909268 R909290 R909292 R909308 R909309 R920394 R920395 R920396 R920397 R920398 R920399 R920404 R920405 R920406 R920407 R920408 R920410 R920411 R925745 R926238 R926752 R926753 R926754 R926755 R926756 R926757 R926759 R926760 R926761 R926762 R926763 R926794 R926826 R926827 R926828 R926829 R926830 R926831 R926832 R926833 R926834 R926835 R926836 R926837 R926838 R926839 R926840 R926841 R926842 R926843 R926844 R926845 R926846 R926847 R926848 R926851 R926855 R926856 R926857 R926859 R926860 R926862 R926863 R926866 R926870 R926871 R926874 R926879 R926880 R926881 R926883 R926885 R926886 R926887 R926890 R926891 R926892 R926893 R926894 R926895 R926896 R926897 R926898 R926899 R926900 R926902 R926903 R926904 R926905 R926906 R926907 R926908 R926909 R926913 R926914 R926915 R926916 R926917 R926918 R926919 R926922 R926923 R926925 R926926 R926927 R926928 R926929 R926930 R926931 R926932 R926933 R926934 R926935 R926936 R926937 R926938 R926939 R926940 R926941 R926942 R926943 R926944 R926945 R926946 R926947 R926948 R926949 R926950 R926951 R926953 R926954 R926955 R926956 R927016 R927017 R927018 R927019 R927020 R927023 R935221 R935222 R935223 R935224 R935225 R935237 R935238 R935239 R935240 R935242 R935248 R935249 R935250 R935251 R935252 R935253 R935255 R935256 R935258 R935259 R935261 R935262 R935263 R935264 R935266 R935267 R935268 R935269 R935271 R935276 R935277 R935278 R935279 R935280 R935281 R935286 R935287 R935288 R935289 R935290 R935291 R935292 R935293 R935294 R935295 R935296 R935297 R935298 R935299 R935300 R935301 R935302 R935303 R935304 R935305 R935306 R935307 R935308 R935309 R935310 R935320 R935321 R935322 R935323 R935324 R935336 R935337 R935338 R935339 R935340 R935351 R935352 R935353 R935354 R935355 R935356 R935357 R935358 R935359 R935360 R935361 R935362 R935363 R935364 R935365 R935366 R935367 R940079 R940110 R940299 R940300 R940301 R940304 R940306 R940307 R940308 R940309 R940311 R940312 R940314 R940316 R940317 R940318 R940319 R940321 R940323 R940337 R940338 R921303 R940345 R940346 R940347 R940348 R940349 R940350 R940351 R940352 R940353 R940354 R945236 R945237 R945242 R945263 R921304 R945299 R950244 R950245 R950246 R950247 R950261 R950262 R950263 R950264 R950265 R950266 R950267 R950290 R950291 R950293 R950294 R950295 R950296 R950344 R950345 R950346 R950347 R950348 R950349 R950356 R950368 R950371 R950372 R950373 R950374 R950376 R950377 R950378 R950379 R950380 R950381 R950382 R950383 R950385 R950386 R950388 R950389 R950391 R950392 R950393 R945028 R935241 R940298 R940302 R940303 R940305 R935260 R909258 R940313 R940315 R935275 R940320 R940322 R926910 R926911 R926912 R926853 R926852 R926854 R926920 R926921 R926924 R926858 R926861 R945298 R940328 R926869 R926873 R926875 R926876 R926877 R940336 R926878 R926882 R926884 R926889 R920400 R920401 R920402 R920403 R940342 R920409 R940344 R926888 R926758 R927024 R927025 R927026 R927027 R927028 R927029 R927030 R927031 R927032 R927035 R927036 R927037 R927038 R927039 R927040 R927041 R927042 R935270 R935368 R935369 R935370 R935371 R935372 R935373 R935374 R935375 R935376 R935377 R935378 R935379 R935380

TABLE 1B CHMC BMMC BMMC BMMC CHMC anti- Ionomycin anti-IgE anti-IgE anti-IgE Compound IgE Tryptase Tryptase Hexos. TNF-alpha IL-6 R908580 R908586 9999 R908587 9999 R908591 0.075 R908592 0.05 R908946 0.51 9999 R908947 0.496 9999 R908950 0.074 47.5 R908951 0.085 5.48 R908952 0.08 6.07 R908953 0.084 R908954 0.084 9999 R908955 0.293 R908956 0.34 R909310 0.207 9999 R909312 1.759 9999 R909313 0.663 9999 R909314 0.293 9999 R909316 0.2 9999 R909317 0.0287 9999 0.002 0.007 0.006 R909318 1.02 9999 R909319 0.225 9999 R909320 0.29 9999 R909321 0.163 30 R909322 0.225 9999 0.24 0.14 0.1 R909323 9999 9999 R926957 1.519 9999 R926958 0.353 9999 R926959 0.3 9999 R926960 0.399 9999 R926961 1.2 9999 R926962 0.205 9999 R926963 0.155 9999 R926964 0.368 9999 R926965 9999 9999 9999 R926966 0.539 9999 R926967 0.259 9999 R926968 0.249 R926969 0.359 9999 R926970 0.06 9999 R926971 0.034 9999 R926972 5.29 9999 R926973 0.284 R926974 0.293 R926975 0.421 30.2 R926976 0.305 8.3 0.59 0.11 0.25 R926977 0.0359 9999 R926978 0.995 18 R926979 0.109 23.5 R926980 0.68 5.49 R926981 0.137 9999 R926982 0.12 9999 R926983 0.195 9999 R926984 0.167 9999 R926985 0.14 4.13 R926986 0.345 R926987 10 R926989 0.199 R926990 11.3 R926991 0.436 R926992 8888 R926993 0.689 R926994 0.061 R926995 9.565 9999 R927004 0.413 R927005 1.158 R927006 2.142 R927007 5.739 R927008 1.123 R927009 4.933 R927010 5.006 R927011 0.464 R927012 3.658 R927013 5.171 R927014 0.655 R927015 9999 9999 R927043 0.45 9999 R927044 9999 4.28 R927045 0.535 9999 R927046 9999 2.4 R927047 0.168 9999 R927048 0.05 9999 R927049 0.11 9999 R927050 0.073 3.29 0.103 0.019 0.011 R927051 0.024 12.6 R927052 0.678 R927053 0.671 R927054 9999 R927055 9999 R927056 0.144 1.58 R927057 0.37 R927058 12.2 R927059 0.291 R927060 0.222 5.17 R927061 0.126 4.72 R927062 15.4 9999 R927063 0.849 9999 R927064 0.212 7.24 0.005 1.92 0.819 R927065 0.235 9999 R927066 0.283 15.3 R927067 0.625 22.5 R927068 0.89 R927069 0.076 13 1.35 0.93 1.09 R927070 0.054 5.24 R927071 0.067 R927072 0.064 R927073 0.0668 R927074 0.072 1.38 R927075 0.057 15.2 R927076 0.071 R927077 0.284 8.8 R927078 0.245 R927079 0.599 R927080 0.204 R927081 2.27 9999 R927082 0.256 9999 R927083 0.316 19 R927084 0.466 9999 R927085 7.43 9999 R927086 0.286 9999 R927087 0.436 9999 R927088 0.117 9999 R927089 0.144 9999 R927090 0.102 9999 R927091 0.27 9999 R927092 0.377 9999 R927093 0.303 9999 R927094 9999 9999 R927096 0.402 9999 R927097 0.163 0.847 R927098 1.53 9999 R927099 9999 9999 R927100 6.199 9999 R927117 0.614 9999 R927118 0.065 3.49 R927119 1.162 R927120 1.018 R927121 0.389 R927122 0.328 R927123 0.087 R927124 0.415 R927125 0.255 R927126 5.167 R927127 9999 R927128 1.893 R927129 1.219 R927130 1.586 R927131 1.473 R927132 2.756 R927133 0.536 R927134 1.286 R927135 0.568 R927136 0.945 R927137 9999.000 R927138 0.463 R927139 9999.000 R927140 4.823 R927141 9999 R927142 5.000 R927143 3.998 R927144 2.273 R927145 5.022 R927146 1.309 R927147 5.088 R927148 0.097 R927149 0.355 R927150 0.708 R927151 0.408 R927152 4.864 R927153 9999.000 R927154 4.978 R927155 8888.000 R927156 2.779 R927157 0.072 R927158 2.284 R927159 4.830 R927160 8888.000 R927162 5.646 R927163 1.827 R931930 0.361 R931931 1.817 R931932 0.511 R931933 0.580 R931934 9999.000 R931935 4.706 R931936 0.957 R931936 9999 R931937 9999.000 R931938 0.542 R931939 0.415 R931940 1.069 R931941 0.494 R931942 5.665 R931943 9999.000 R931944 0.285 R931945 9999.000 R931946 5.594 9999 R931947 2.700 9999 R931948 0.197 R931949 0.033 R931950 1.243 R931951 0.017 R931952 0.166 R935381 9999 7.74 R935382 9999 0.2 R935383 0.146 9999 R935384 9999 9999 R935385 9999 0.217 R935386 0.291 R935389 0.877 R935390 0.544 R935391 0.212 9999 0.25 0.19 0.55 R935392 0.204 9999 R935393 8888 9999 2.44 1.47 0.52 R935394 9999 R935395 0.276 R935396 2.58 R935398 8888 R935399 0.909 R935400 0.502 R935401 0.51 R935402 0.216 R935403 0.821 R935404 0.581 R935405 0.389 R935406 1.17 R935407 0.393 R935408 0.137 9.94 R935409 1.17 R935410 0.417 R935411 9999 R935413 0.085 9999 R935412 0.696 R935414 0.204 R935415 0.237 R935416 0.166 R935417 0.417 R935418 0.228 9999 R935419 0.23 R935420 0.561 R935421 2.89 R935422 0.326 R935423 0.167 R935424 0.628 R935425 8888 R935426 9999 R935427 8888 R935428 1.272 R935429 0.036 9999 R935430 0.028 9.3 R935431 0.124 R935432 0.036 8.5 R935433 0.106 16.2 R935434 0.308 R935435 0.337 R935436 0.058 R935437 0.082 R935438 0.414 23 R935439 R935440 0.176 88 R935441 0.586 R935442 0.701 R935443 8888 R935444 0.429 9999 R935445 0.184 11 R935446 0.395 9999 R935447 0.511 4.7 R935448 0.111 4.3 R935449 0.372 7.8 R935450 0.494 9999 R935451 9999 9999 R935452 0.213 9999 R935453 0.15 9999 R935458 8888 9999 R935459 0.343 4.7 R935460 0.748 15.6 R935461 0.134 5.03 R935462 0.364 9999 R935463 0.176 9999 R935464 22.4 9999 R935465 0.019 4.22 R935466 0.284 R935467 0.352 R935468 0.705 5.37 R935469 0.039 3.79 R935469 0.056 R935470 0.804 4.90 R935471 0.481 R935472 1.056 R935473 0.057 R935474 0.474 R935475 0.516 R935476 0.639 R935477 0.097 R935478 1.700 R935479 1.355 R935480 4.576 R935481 0.114 R935482 0.743 R935483 0.601 R935484 1.252 R935485 0.231 R935486 1.845 R935487 3.224 R935488 4.443 R935489 0.185 R935490 1.474 R935491 6.873 R935492 26.130 R935493 0.385 R935494 3.063 R935495 1.112 R935496 1.952 R935497 0.097 R935498 1.016 R935499 1.207 R935500 1.588 R935501 0.305 R935502 1.466 R935503 0.400 R935504 2.777 R935505 0.038 R935506 0.375 R935507 0.473 R935508 0.967 R935509 0.086 R935510 0.897 R935511 1.165 R935512 2.098 R935513 0.106 R935514 1.662 R935515 2.661 R935516 2.800 R935517 0.548 R935518 2.963 R935519 0.074 R935520 0.001 R935521 0.186 R935522 1.236 R935523 0.001 R935524 0.249 R935525 1.564 R935526 9.126 R935527 0.557 R935528 3.332 R935529 0.245 R935529 9999 R935531 9999 R935531 0.871 R935532 9999 R935532 0.110 R935533 9999 R935533 0.219 R935534 0.398 5.218 R940355 99 9999 R940356 7.21 9999 R940358 0.03 4.3 R940361 0.047 2.2 0.06 0.07 0.1 R940363 0.048 9999 R940364 0.046 9999 R940365 8888 9999 R940366 0.037 40 0.03 0.005 0.01 R940367 0.117 14.1 R940368 0.025 1.58 R940369 0.023 9999 R940370 S 0.059 — R940371 0.316 R940372 0.094 R940373 8888 R940380 0.042 R940381 8888 R940382 0.104 R940383 0.064 R940384 1.32 R940385 0.033 R940386 3.42 R940387 1.19 R940388 0.049 R940389 0.06 R940390 9999 9999 R940391 0.261 R940392 0.145 R940393 5.26 R940394 16.5353 R940395 9999 R940396 22.7164 R940397 3.7 R940399 0.051 R940400 0.103 R940401 0.125 R940402 8888 R945356 1.17 9999 R945357 9999 9999 R945358 9999 9999 R945360 1.37 9999 R945361 2.36 9999 R945362 1.57 9999 R945363 0.687 9999 R945364 1.002 9999 R945365 0.257 9999 R945366 0.112 9999 R945367 9999 1.29 R945368 9999 1.71 R945369 9999 1.27 R945370 0.522 9999 R945371 0.713 9999 R945372 9999 0.923 R945373 9999 R945374 9999 R945375 9999 R945376 9999 R945377 1.12 R945378 0.754 R945379 9999 R945380 9999 R945381 9999 R945382 9999 R945383 0.985 R945384 0.913 R945385 1.1 R945386 1.39 R945387 1.12 R945389 0.0748 9999 R945390 0.118 9999 R945391 0.094 9999 R945392 0.085 9999 R945393 1.34 21.7 R945394 1.24 5.61 R945395 1.14 9999 R945396 2.24 R945397 0.928 R945398 7 R945399 0.163 9999 R945400 9999 R945401 8888 9999 R945402 0.112 R945403 1.7 R945404 0.103 R945405 0.131 R945406 8888 R945407 8888 R945408 9999 R945409 9999 R945410 9999 R945411 2.86 R945412 0.095 R945413 1.698 R945414 0.038 R945415 0.046 R945416 0.053 R945417 2.52082 9999 R945418 8888 9999 R945419 0.125 R945420 0.436 R945421 0.371 R945422 0.092 R945423 0.145 R945424 0.188 R945426 0.256 R945427 0.279 R945432 0.049 R945433 0.276 R945434 8888 R945439 8888 R945440 8888 R945443 0.081 9999 R945444 0.043 9999 R945454 20.6 9999 R945455 8888 9999 R945456 8888 R945457 0.188 R945458 8888 R945459 0.038 R945460 1.184 R945461 0.803 R945462 1.722 R945463 0.722 R945464 0.943 R945465 1.960 R945466 1.885 R945467 1.169 R945470 0.862 R945471 0.035 R945472 0.094 R945473 0.104 R945474 0.104 R945475 0.046 R945476 0.293 R945477 0.363 R945478 0.153 R945479 0.272 R945480 0.199 R945485 0.850 R945486 0.588 R945491 0.465 R945492 0.079 R945493 0.069 R945498 0.001 9999 R950405 1.36 9999 R950406 9999 9999 R950407 9999 9999 R950408 9999 4.82 R950409 9999 3.24 R950410 9999 9999 R950411 9999 4 R950412 0.301 R950413 9999 9999 R950414 9999 9999 R950415 5.19 16.3 R950416 2.27 R950417 2.16 9999 R950418 1.67 9.09 R950419 3.26 9999 R950420 0.114 9999 R950421 0.157 9999 R950422 0.475 6.53 R950423 0.05 9999 R950424 0.236 4.28 R950425 1.15 R950426 0.142 30 R950427 1.9 R950428 0.123 21 R950429 3.969 R950430 0.239 R950432 2.42 R950433 9999 R950434 1.16 R950436 5.53 R950437 0.811 R950438 0.888 R950439 9999 R950440 10.47 R950441 9999 R950442 9999 9999 R950443 9999 9999 R950444 1.73 R950445 0.379 R950446 0.148 R950447 1.41999 9999 R950448 1.08228 36 R950449 0.668 R950450 1.09 R950451 0.07 R950452 0.101 R950453 8888 9999 R950454 8.6351 9999 R950455 0.217 R950456 3.78374 4.4 R950457 3.08825 9999 R950458 1.32355 12 R950459 0.632 R950460 0.177 R950461 0.142 R950462 9999 R950463 2.46 R950464 0.244 R950465 0.351 R950469 9999 9999 R950470 16.1729 9999 R950471 50.5397 9999 R950472 6.95156 9999 R950493 1.89 R950494 9999 R950495 2.2 R950496 12.4 R950497 8888 R950498 9999 R950499 0.199 R950500 1.694 R950501 0.430 R950502 2.496 R950503 2.085 R950504 1.275 R950505 9999.000 R950506 9999.000 R950507 0.106 R950508 44.555 9999 R950509 0.112 R950510 0.093 R950511 9999.000 R950512 6.611 R950513 7.049 R950514 0.244 R950515 0.031 R950516 0.025 R950518 1.405 R950519 6.488 R950520 0.397 4.513 R950521 0.145 5.814 R950522 0.123 9999 R950523 0.084 7.728 R950524 0.224 5.963 R950525 0.292 14.819

TABLE 2 High Density Toxicity Toxicity CHMC CHMC CHMC CHMC CHMC CHMC Toxicity Jurkat Toxicity BJAB high density high density high density high density high density high density Jurkat Cell BJAB Cell hexos tryptase histamine LTC4 TNF-alpha IL-13 Light Scat. Titer Glo Light Scat. Titer Glo R008951 R008952 R008953 R008955 R008956 R008958 R067934 R067963 R070153 R070791 R081166 R088814 R088815 R091880 R092788 9999 9999 R909241 3.736 R921219 0.124 0.121 0.162 0.034 0.190 0.175 >10 >10 R925775 9999 9999 R925778 9999 9999 R925779 >10 9999 R925797 >10 9999 R926108 >10 >10 R926109 0.783 0.906 1.827 0.808 1.504 1.664 >10 9999 R926110 >10 >10 R921218 0.464 0.647 0.463 0.695 1.752 2.0776 >10 >10 R926113 1.448 1.649 1.848 0.468 5.678 3.569 >10 >10 R926146 9999 9999 R926210 >10 9999 R926240 10 9999 R926248 >10 9999 R926249 >10 9999 R926253 9999 9999 R926256 >10 9999 R926258 9999 9999 R926387 >10 9999 R926395 >10 9999 R926396 >10 9999 R926411 8.5 >10 R926486 1.088 1.313 1.928 0.834 0.455 R926488 0.521 0.623 0.792 0.201 2.443 1.012 R926493 0.889 1.093 1.324 0.474 >2 >4.33 R926494 0.640 >2 9999 0.326 9999 R926495 0.100 0.235 0.066 0.241 0.362 0.449 >10 >10 R926496 0.429 0.533 0.809 0.414 0.622 R926497 1.106 1.234 1.333 1.876 9999 R926501 >2 >2 9999 9999 9999 >4.33 >4.33 R926502 >2 >2 >2 1.807 >2 1.513 R926505 4.199 R926508 0.170 0.434 0.105 0.505 0.763 >10 >10 R926510 0.921 1.115 1.667 0.417 0.686 2.77 R926511 1.183 1.474 1.73 1.307 >2 >4.33 >4.33 R926614 >10 >10 >10 6.442 R926696 <1.1 <1.1 <1.1 <1.1 <1.1 1.773 >5.0 R926699 <1.1 <1.1 1.44 <1.1 <1.1 1.294 R926700 <1.1 <1.1 <1.1 <1.1 <1.1 2.053 R926703 1.512 1.947 >2 0.724 >2 R926704 >2 9999 9999 9999 9999 R926705 1.007 1.256 0.641 0.494 9999 R926706 >2 9999 9999 1.491 9999 R926742 0.104 0.217 0.080 0.385 0.667 9 >10 R926745 >10 >10 R926780 >5.0 R926782 >4.33 >4.33 R935075 0.647 1.212 0.443 <0.22 >2 >4.33 >4.33 R935154 >4.33 R935156 4.054 R940216 <1.1 <1.1 1.176 <1.1 3.188 3.006 R940233 0.577 0.642 0.586 0.118 2.247 1.781 >4.33 >4.33 R945032 0.357 0.458 0.439 0.0929 1.082 0.291 R945033 8.151 8.868 >10 5.983 R945071 <1.1 <1.1 <1.1 <1.1 <1.1 <1.1 R945128 1.279 1.749 0.547 0.729 >2 ND R945140 0.994 1.112 1.551 1.714 9999 R945142 >2 >2 9999 >2 9999 R945150 >4.33 >4.33 R921302 0.682 0.795 1.588 0.514 1.173 1.672 R950141 0.567 0.618 0.627 0.201 1.059 0.798 R950207 >4.33

7.7 The 2,4-Pyrimidinediamine Compounds of the Invention Selectively Inhibit the Upstream IgE Receptor Cascade

To confirm that many of the 2,4-pyrimidinediamine compounds of the invention exert their inhibitory activity by blocking or inhibiting the early IgE receptor signal transduction cascade, several of the compounds were tested in cellular assays for ionomycin-induced degranulation, as described below.

7.7.1 CHMC Low Cell Density Ionomycin Activation Tryptase Assay

Assays for ionomycin-induced mast cell degranulation were carried out as described for the CHMC Low Density IgE Activation assays (Section 7.5.2, supra), with the exception that during the 1 hour incubation, 6× ionomycin solution [5 mM ionomycin (Sigma 1-0634) in MeOH (stock) diluted 1:416.7 in MT buffer (2 μM final)] was prepared and cells were stimulated by adding 25 μl of the 6× ionomycin solution to the appropriate plates.

7.7.2 Basophil Ionomycin Activation Histamine Release Assay

Assays for ionomycin-induced basophil cell degranulation were carried out as described for the Basophil IgE or Dustmite Activation Assay (Section 7.5.5, supra), with the exception that following incubation with compound, cells were stimulated with 20 μl of 2 μM ionomycin.

7.7.3 Results

The results of the ionomycin-induced degranulation assays, reported as IC₅₀ values (in μM) are provided in TABLE 1, supra. Of the active compounds tested (i.e., those that inhibit IgE-induced degranulation), the vast majority do not inhibit ionomycin-induced degranulation, confirming that these active compounds selectively inhibit the early (or upstream) IgE receptor signal transduction cascade.

These results were confirmed for certain compounds by measuring anti-IgE-induced and ionomycin-induced calcium ion flux in CHMC cells. In these Ca²⁺ flux tests, 10 μM R921218 and 10 μM R902420 inhibited anti-IgE-induced Ca²⁺ flux, but had no effect on ionomycin-induced Ca²⁺ flux (See FIG. 4).

7.8 The Inhibitory Effect of the 2,4-Pyrimidinediamine Compounds of the Invention is Immediate

To test the immediacy of their inhibitory effect, certain 2,4-pyrimidinediamines of the invention were added simultaneously with anti-IgE antibody activator in the cellular assays described above. All compounds tested blocked IgE-induced degranulation of CHMC cells to the same extent as observed when the compounds were pre-incubated with CHMC cells for 10 or 30 min. prior to receptor cross-linking.

7.9 Kinetics of Pharmacological Activity In Vitro

Compounds R921218, R921302, R921219, R926240, R940277, R926742, R926495, R909243 and R926782 were tested in washout experiments. In the experiments, CHMC cells were either activated immediately with anti-IgE antibody in the presence of 1.25 μM compound (time zero), or the compound was washed out followed by activation with anti-IgE antibody at 30, 60 or 120 min. The inhibitory activity of these compounds was greatly diminished 30 min. after compound removal, indicating that constant exposure of mast cells to these compounds is required for maximal inhibition of degranulation The other compounds tested yielded similar results.

7.10 Toxicity T- and B-Cells

The ability of the compounds of the invention to exert their inhibitory activity without being toxic to cells of the immune system was demonstrated in cellular assays with B- and T-cells. The protocols for the assays are provided below.

7.10.1 Jurkat (T-Cell) Toxicity

Dilute Jurkat cells to 2×10⁵ cells/ml in complete RPMI (10% heat-inactivated fetal bovine serum) media and incubate at 37° C., 5% CO₂ for 18 hours. Add 65 ul cells at 7.7×10⁵ cells/ml to a 96-well V-bottom plate (TC-treated, Costar) containing 65 ul 2× compound (final vehicle concentration is 0.5% DMSO, 1.5% MeOH). Mix, incubate plates for 18-24 hr at 37° C., 5% CO₂. Toxicity was assessed by flow cytometric analysis of cellular light scatter

7.10.2 BJAB (B-Cell) Toxicity

The B-cell line BJAB was cultured in log phase in RPMI1640+10% heat-inactivated fetal bovine serum, 1× L-glutamine, 1× penicillin, 1× streptavidin and 1× beta-mercaptoethanol at 37° C., 5% CO2. First, BJABs were harvested, spun and resuspended in culture medium to a concentration of 7.7×10⁵ cells/mL. 65 uL cells were mixed with 65 uL compound, in duplicate and in the presence of 0.1% DMSO in a V-bottomed 96-well tissue culture plate. Cells were incubated with compound at various dilutions at 37° C., 5% CO₂. Toxicity was assessed by flow cytometric analysis of cellular light scatter.

7.10.3 Toxicity Cell Titer Glo Assay

Seed 50 μl cells (1×10⁶/ml) into each well containing 50 μl compound. The final vehicle concentration is 0.5% DMSO, 1.5% MeOH. Shake plates for 1 minute to mix cells and compound. Incubate plates at 37° C. (5% CO₂) for 18 hours. Next day, harvest 50 μl cells from each well, add to 50 μl Cell Titer Glo reagent (Invitrogen). Shake plates for 1 minute. Read on luminometer.

7.10.4 Results

The results of the T- and B-cell toxicity assays, reported as IC₅₀ values (in μM), are presented in TABLE 2, supra. With a few exceptions (see TABLE 1), all compounds tested were non-toxic to both B- and T-cells at effective inhibitory concentrations. Assays performed with primary B-cells yielded similar results.

7.11 The 2,4-Pyrimidine Compounds are Tolerated in Animals

The ability of the compounds of the invention to exert their inhibitory activity at doses below those exhibiting toxicity in animals was demonstrated with compounds R921218, R921219 and R921302.

7.11.1 R921218

R921218 was studied in an extensive program of non-clinical safety studies that concluded this agent to be well tolerated in both rodents and non-rodents. To summarize the outcome of toxicology/non-clinical safety testing with R921218; this agent produced no dose limiting toxicity by the intranasal route of administration in non-rodents (rabbits and primates) or by the oral route of administration in rodents (mice and rats) during 14-day repeat-dose toxicity studies at doses many fold above the anticipated dose expected to produce efficacy in man. There were no adverse findings in a core safety pharmacology battery of cardiovascular, respiratory and/or central nervous system function. There was no evidence for mutagenic or clastogenic potential in genetic toxicology testing nor were there untoward effects after exposure to skin and eyes. A short discussion of key toxicology studies is provided.

A 14-day repeat-dose intranasal toxicity study in Cynomolgus monkeys was performed at doses of 2.1, 4.5 or 6.3 mg/kg/day. In life parameters included: clinical observations, body weights, food consumption, ophthalmology, blood pressure, electrocardiography, hematology, clinical chemistry, urinalysis, immunotoxicological assessment, gross necropsy, organ weights, toxicokinetic assessments and histopathology (including the nasal cavity). There were no adverse findings attributed to R921218 in any study parameter and the NOAEL (no observed adverse effect level) was considered 6.3 mg/kg/day.

A 14-day repeat-dose intranasal toxicity study in New Zealand White rabbits was performed at doses of 1.7, 3.4 or 5.0 mg/kg/day. In life parameters included: clinical observations, body weights, food consumption, ophthalmology, hematology, clinical chemistry, gross necropsy, organ weights, toxicokinetic assessments and histopathology (including the nasal cavity). There were no adverse findings attributed to R921218 in any study parameter and the NOAEL (no observed adverse effect level) was considered 5.0 mg/kg/day.

7.11.2 R921219

In pilot dose finding studies a single dose oral dose of 600 mg/kg was considered a NOEL (no observed effect level) while multiple (7-day) doses of 200 mg/kg/day and above were not tolerated.

In the in vitro Salmonella-Escherichia coli/Mammalian-Microsome Reverse Mutation Assay (Ames test), R921219 was found to test positive in tester strain TA1537, with and without metabolic activation, confirming the results of an earlier study. R921219 was not found to adversely affect any of the other 4 tester strains. R921219 was not found to possess clastogenic potential when studied in an in vitro chromosomal aberration assay.

7.11.3 R921302

Several non-GLP pilot toxicity studies have been conducted in rodents. In the mouse an oral dose of 1000 mg/kg was tolerated for up to 7-days. In a 14-day oral toxicity study in the mouse was conducted with doses of 100, 300 and 1000 mg/kg. A dose of 1000 mg/kg was not tolerated, while a dose of 300 mg/kg promoted evidence for histopathological changes in the vulva. A dose of 100 mg/kg was considered the NOAEL (no observed adverse effect level) in the study. A 28-day oral toxicity study in the mouse was conducted at doses of 100 mg/kg q.d., 100 mg/kg b.i.d., 300 mg/kg q.d. and 300 mg/kg b.i.d. R921302 was not tolerated at 300 mg/kg q.d. or b.i.d. The lower doses (100 mg/kg q.d. or b.i.d.) appeared to be well tolerated (results of clinical and histopathology are not yet known). In the rat oral doses of 50, 150 and 300 mg/kg given for 32 days appeared to be well tolerated (results of clinical and histopathology are not yet known).

In the in vitro Salmonella-Escherichia coli/Mammalian-Microsome Reverse Mutation Assay (Ames test), R921302 was found to test positive in tester strain TA98 with S9 and TA1537, with and without metabolic activation. R921302 was not found to adversely affect any of the other 3 tester strains. R921302 was not clastogenic when assessed in an in vitro chromosomal aberration assay.

7.12 The 2,4-Pyrimidinediamine Compounds are Orally Bioavailable

Over 50 2,4-pyrimidinediamine compounds of the invention were tested for oral bioavailability. For the study, compounds were dissolved in various vehicles (e.g. PEG 400 solution and CMC suspension) for intravenous and oral dosing in the rats. Following administration of the drug, plasma samples were obtained and extracted. The plasma concentrations of the compounds were determined by high performance liquid chromatography/tandem mass spectrometry (LC/MS/MS) methods. Pharmacokinetic analyses were performed based on the plasma concentration data. The pharmacokinetic parameters of interest include Clearance (CL), Volume of distribution at steady-state (Vss), terminal half-life (t 4 and oral bioavailability (% F).

These pharmacokinetic studies indicate that many of the 2,4-pyrimidinediamine compounds are orally available, with % F up to approximately 50% (in the range of 0-50%). The half-lives ranged from 0.5 to 3 hr. In particular, Compounds R940350, R935372, R935193, R927050 and R935391 exhibited good oral bioavailabilities and half-lives in rats. Thus, these studies confirm that these 2,4-pyrimidinediamine compounds are suitable for oral administration.

7.13 The Compounds are Effective for the Treatment of Allergies

The in vivo efficacy of compounds R926109, R921218, R921219, R921302, R926495, R926508, R926742, R926745 and R945150 towards allergies was evaluated in the mouse model of passive cutaneous anaphylaxis (PCA). This model provides a direct measure of IgE-induced degranulation of tissue mast cells. In this model, IgE primed animals are exposed to an allergen challenge, and the change in permeability of dermal vasculature that results from histamine release from mast cells is measured by change in the amount of dye leakage into surrounding tissue Inhibition of mediator release by compounds that modulate mast cell degranulation is easily measured by extracting the dye from the tissue.

7.13.1 Study Protocol and Results

In the PCA assay mice are passively sensitized by intradermal injection with anti-dinitrophenol (DNP) IgE antibodies (Day −1). At predetermined times animals are treated with the test agent (Day 0). The modulatory effect of the agent on cutaneous mast cell degranulation is measured following intravenous injection of DNP conjugated to human serum albumin (HSA-DNP), together with Evans blue dye. The resulting cross-linking of the IgE receptor and subsequent mast cell degranulation-induced increase in vascular permeability is determined by measuring the amount of dye extravasation into the tissue. Dye is extracted from the tissue by formamide, and the absorbance of this extract is read at 620 nm. The inhibitory effect of drug treatment is reported as the percent inhibition compared to vehicle treatment, that is, the percent reduction in A₆₂₀.

Two compounds have been tested as positive controls: the histamine antagonist diphenhydramine and the serotonin antagonist cyproheptadine. Both mediators (histamine and serotonin) are released upon IgE-mediated degranulation from the mouse mast cell. Both reference compounds inhibit the PCA response; cyproheptadine was used routinely in subsequent experiments. Cyproheptadine reproducibly inhibited the PCA response by 61%+/−4% (8 mg/kg, i.p., 30 minutes pretreatment time, n=23 experiments).

7.13.1.1 Results

A dose-dependent inhibition of the FcεR-mediated vascular leakage was observed with increasing doses of R921218, R926109, R921219 and RR921302. These compounds were administered either in a solution formulation (67% PEG/33% citrate buffer) or an aqueous suspension (1.5% Avicel). These results demonstrate the strong correlation between compound plasma levels, in vivo efficacy, and in vitro potency. The most potent compound, R921219, was active with circulating exposure levels of approximately 10 mg/ml (68% inhibition at a dose level of 100 mg/kg) compared with R921302, a relatively less potent molecule, which reduced plasma extravasation by 42% at a dose level of 100 mg/kg. Further, the length of exposure to circulating compound was reflected in the duration of inhibitory activity. R921302, determined to be the most metabolically stable compound in pharmacokinetics studies, inhibited the vascular permeability for 1-2 hours prior to antigen-induced receptor signaling, where after the efficacy began to decrease. These data are summarized in TABLE 3 and TABLE 4.

TABLE 3 Efficacy of R921218, R926109, R921219 and R921302 in the PCA Assay Pre- treat- ment Plasma Com- time Dose % level pound Route Vehicle (min) (mg/kg) Inhibition (μg/ml) R921218 PO 67% PEG/33% 10 50 7 3 citrate buffer 100 11 4 200 50 18 R926109 PO 67% PEG/33% 15 50 22 N.D. citrate buffer 100 32 200 48 R921219 PO 1.5% 15 30 25 0.4 Avicel/water 100 68 4 300 92 11 R921302 PO 1.5% 60 50 35 25 Avicel/water 100 42 38 150 56 64 200 93 105

TABLE 4 Duration of action of R921219 and R921302 in the PCA Assay Pre- Plasma Dose treatment % level Compound Route Vehicle (mg/kg) time (min) Inhibition (μg/ml) RR921302 PO 1.5% 200 30 89 88 Avicel/ 60 83 53 water 120 82 61 240 37 8 Similar in vivo activity was observed with compounds R926495, R926508, R926742, R926745 and R926150, which were able to inhibit the PCA response after administration by the oral route in a PEG-based formulation (data not shown).

7.14 The Compounds are Effective in the Treatment of Asthma

The efficacy of compounds R921218, R921302, R926495, R926508, R926742 and R921219 in the treatment of asthma was demonstrated in the sheep model of allergic asthma. Sheep develop bronchoconstriction within minutes of exposure to inhaled antigen (Ascaris suum), with maximal airflow obstruction during the early allergic response (EAR). Release of preformed mast cell mediators is likely responsible for this early phase of airflow obstruction. In addition to the EAR, the sheep model allows us to evaluate the effect of our compounds on the late asthmatic reaction (LAR) and non-specific airway hyperresponsiveness (AHR), which occur as a result of topical or local administration of allergen to the airway. In the sheep, AHR develops a few hours following antigen challenge, and can persist for up to 2 weeks. The results described below demonstrate the potential of the tested compounds to inhibit a cascade of events that may be a result of release of cytokines from the mast cell.

7.14.1 Study Protocol

In the sheep model of allergic asthma, sheep are administered aerosols of test article via an endotracheal tube, followed by an aerosol challenge with antigen extracted from the roundworm, Ascaris suum, to which the sheep are naturally allergic. Allergen challenge leads to direct bronchoconstriction (both EAR and LAR) and a persistent non-specific AHR. These three characteristics are similar to those seen in human allergic asthmatics. The activity of the test agent is determined by changes in the lung resistance (R_(L)), which is calculated from measurements of transpulmonary pressure, flow, and respiratory volume. The historical control data obtained from the same sheep following saline treatment compared with an allergen challenge show that a sharp increase of R_(L) occurs during the EAR and persists for approximately 2-3 hours following allergen challenge. The LAR is a less pronounced increase in R_(L), which starts approximately 5-6 hours following allergen challenge and is resolved by 8 hours post-challenge. Twenty-four hours after the challenge, a dose response to carbachol is measured to determine the AHR, which is expressed as the dose of carbachol required to increase R_(L) by 400% over baseline. (This measurement is referred to as the provocative concentration of carbachol that elicits a 400% increase in RL over baseline (PC₄₀₀). The data are compared to historical control data for the same individual when administered a saline control aerosol and challenged with Ascaris suum.

7.14.2 Result

All the compounds tested showed inhibitory effects in the LAR and the AHR, and several of these agents inhibited the EAR as well. The optimal response for each compound in a series of studies to evaluate activity at several pretreatment times and using several different solution and suspension formulations are shown in TABLE 5. The efficacy of R921218 on the EAR appeared to be dependent on the formulation, with the greatest effect seen at 30 mg/sheep administered as a solution aerosol in 10% ethanol. R926495, R926742, R926508 and R921219, administered in four different sheep at 45 mg/sheep in an aqueous suspension 60 minutes prior to allergen challenge, demonstrate that the LAR and AHR is blocked. In addition to these late parameters, the EAR was greatly reduced by treatment with R921219, R926508 or R926495. The efficacy of RR921302 was investigated using a 45% PEG400/55% citrate buffer vehicle. Under these conditions, R921302, administered at 30 mg/sheep 60 minutes prior to challenge, blocked the LAR and AHR, and EAR was unaffected.

These data clearly demonstrate that these compounds are able to block the asthmatic responses in allergic sheep. All compounds inhibited the AHR and LAR significantly when compared to the historical control. The EAR was significantly inhibited by R921219, R926508 and R926495 (54%, 21% and 33% respectively). In contrast, R921218, R921302 and R926742 failed to inhibit the EAR when administered in an aqueous suspension.

TABLE 5 Efficacy Of Exemplary Compounds In A Sheep Model Of Allergic Asthma Pre- treat- Dose ment EAR AHR Com- (mg/ time (% inhi- LAR (% (% inhi- pound sheep) (min) Vehicle bition) inhibition) bition) R921218 30 15 10% ethanol 66 78 101 R926742 45 60 Aqueous −19 87 94 R926495 45 60 suspension 33 85 41 R926508 45 60 21 90 88 R921219 45 60 56 75 90 RR921302 30 60 45% −28 86 82 PEG400/ 55% citrate buffer

7.15 The Compounds are Effective in the Treatment of Asthma

The efficacy of compounds R921304 and R921219 in the treatment of asthma was also demonstrated in a mouse model of allergic asthma.

7.15.1 Study Protocol

Mice are sensitized to ovalbumin (chicken protein) in the presence of an adjuvant (Alum) by the intraperitoneal route on day 0 and day 7. One week later, mice are challenged intranasally with ovalbumin on Days 14, 15 and 16 (more stringent model) or on Day 14 (less stringent model). This sensitization and challenge regimen leads to airway hyperresponsiveness and inflammation in the lungs, which are two dominant characteristics of human allergic asthma. In the mouse model, the in vivo airway responses are measured using a whole body plethysmograph which determines the PENH (enhanced Pause, Buxco Electronics). The PENH is a dimensionless value comprised of the peak inspiratory flow (PIF), peak expiratory flow (PEF), time of inspiration, time of expiration and relaxation time, and is considered a validated parameter of airway responsiveness. Responses to allergen challenge (OVA) are compared with animals challenged with saline only. Twenty-four hours after challenge, mice are exposed to increasing doses of methacholine (muscarinic receptor agonist) which results in smooth muscle contraction. The ovalbumin-challenged mice demonstrate a significant airway hyperresponsiveness to methacholine when compared to the saline challenged mice. In addition, a cellular infiltrate in the airway is observed in ovalbumin challenged mice when compared with the saline challenged mice. This cellular infiltrate is mainly characterized by eosinophils, but a smaller influx of neutrophils and mononuclear cells is also present.

The use of this model for the evaluation of small molecule inhibitors of mast cell degranulation has been validated is several ways. First, using mast cell deficient mice (W/W^(v)) it has been shown that the ovalbumin-induced responses are dependent upon the presence of mast cells. In the mast cell deficient mice, ovalbumin sensitization and challenge did not result in airway hyperresponsiveness and eosinophil influx. Second, the mast cell stabilizer, Cromolyn, was able to block the ovalbumin-induced airway hyperresponsiveness and inflammation (data not shown). The use of this model to evaluate compounds for the treatment of asthmatic responses that may be mediated by mechanisms other than mast cell stablization, is further supported by the inhibitory effect of the steroids, dexamethasone and budesonide, on methacoline-induced bronchocontriction.

7.15.2 Results

The efficacy of R921304 was evaluated by intranasal administration on 10 consecutive days, from Day 7 through Day 16, at a dose level of 20 mg/kg, with the last 3 doses administered 30 minutes prior to either saline or ovalbumin challenge. R921304 was able to inhibit the ovalbumin-induced airway hyperresponsiveness to methacholine when compared to the vehicle treated mice.

In a less stringent protocol, in which the mice were challenged with ovalbumin only once on Day 14, R921219 administered subcutaneously at 70 mg/kg in 67% PEG400/33% citrate buffer 30 minutes prior to saline or ovalbumin challenge, demonstrates that R921219 completely blocked the ovalbumin-induced airway hyperresponsiveness and cellular influx.

These results clearly demonstrate that R921219 and R921304 are efficacious in inhibiting the airway responses in a mouse model of allergic asthma

7.16 2,4-Pyrimidinediamine Compounds Inhibit Phosphorylation of Proteins Downstream of Syk Kinase in Activated Mast Cells

The inhibitory effect of the 2,4-pyrimidinediamine compounds on the phosphorylation of proteins downstream of Syk kinase was tested with compounds R921218, R218219 and R921304 in IgE receptor-activated BMMC cells.

For the assay, BMMC cells were incubated in the presence of varying concentrations of test compound (0.08 μM, 0.4 μM, 2 μM and 10 μM) for 1 hr at 37° C. The cells were then stimulated with anti-IgE antibody as previously described. After 10 min, the cells were lysed and the cellular proteins separated by electrophoresis (SDS PAGE).

Following electrophoresis, the phosphorylation of the proteins indicated in FIGS. 7, 10 and 11A-D were assessed by immunoblot. Antibodies were purchased from Cell Signaling Technology, Beverley, Mass.

Referring to FIGS. 7, 10 and 11A-D, the indicated compounds tested inhibited phosphorylation of proteins downstream of Syk, but not upstream of Syk, in the IgE receptor signaling cascade, confirming both that the compounds inhibit upstream IgE induced degranulation, and that the compounds exert their inhibitory activity by inhibiting Syk kinase.

7.17 2,4-Pyrimidinediamine Compounds Inhibit Syk Kinase in Biochemical Assays

Several 2,4-pyrimidinediamine compounds were tested for the ability to inhibit Syk kinase catalyzed phosphorylation of a peptide substrate in a biochemical fluorescenced polarization assay with isolated Syk kinase. In this experiment, Compounds were diluted to 1% DMSO in kinase buffer (20 mM HEPES, pH 7.4, 5 mM MgCl₂, 2 mM MnCl₂, 1 mM DTT, 0.1 mg/mL acetylated Bovine Gamma Globulin). Compound in 1% DMSO (0.2% DMSO final) was mixed with ATP/substrate solution at room temperature. Syk kinase (Upstate, Lake Placid N.Y.) was added to a final reaction volume of 20 uL, and the reaction was incubated for 30 minutes at room temperature. Final enzyme reaction conditions were 20 mM HEPES, pH 7.4, 5 mM MgCl₂, 2 mM MnCl₂, 1 mM DTT, 0.1 mg/mL acetylated Bovine Gamma Globulin, 0.125 ng Syk, 4 uM ATP, 2.5 uM peptide substrate (biotin-EQEDEPEGDYEEVLE-CONH2, SynPep Corporation). EDTA (10 mM final)/anti-phosphotyrosine antibody (1× final)/fluorescent phosphopeptide tracer (0.5× final) was added in FP Dilution Buffer to stop the reaction for a total volume of 40 uL according to manufacturer's instructions (PanVera Corporation) The plate was incubated for 30 minutes in the dark at room temperature. Plates were read on a Polarion fluorescence polarization plate reader (Tecan). Data were converted to amount of phosphopeptide present using a calibration curve generated by competition with the phosphopeptide competitor provided in the Tyrosine Kinase Assay Kit, Green (PanVera Corporation).

The results of the assay are shown in TABLE 6, below:

TABLE 6 SYK SYK SYK Kinase Kinase Kinase Com- IC50 Com- IC50 IC50 pound (in μM) pound (in μM) Compound (in μM) R908701 0.022 R927060 0.62 R940376 0.067 R908702 0.038 R927061 0.158 R940380 0.029 R908712 0.024 R927064 0.466 R940381 4999.846 R908952 0.041 R927069 0.111 R940382 0.144 R908953 0.017 R927077 0.602 R940384 9999 R908956 1.178 R927078 0.222 R940386 19.49 R909236 2.071 R927080 0.254 R940387 9999 R921219 0.041 R927082 0.312 R940388 0.268 R909268 0.125 R927083 0.449 R940389 0.053 R909309 0.09 R935138 0.229 R940390 9999 R909317 0.008 R935189 0.354 R945071 0.43 R909321 0.104 R935190 0.047 R945140 0.611 R909322 0.141 R935191 0.045 R945142 2.007 R920410 0.187 R935193 0.11 R945144 0.612 R921218 0.254 R935194 0.169 R945157 1.762 R926242 1.81 R935196 0.266 R921304 0.017 R926252 9999 R935198 0.2 R945299 0.022 R926321 5049 R935202 0.035 R945365 0.465 R926500 0.929 R935237 0.046 R945366 0.059 R926501 0.193 R935293 0.047 R945369 1.85 R926502 0.217 R935302 0.027 R945370 1.05 R926505 0.07 R935304 0.042 R945371 1.3 R926508 0.097 R935307 0.057 R945385 2.12 R926562 9999 R935309 0.098 R945389 0.035 R926594 0.771 R935310 0.206 R945390 0.009 R926715 0.534 R935366 0.38 R945391 0.01 R926742 0.076 R935372 0.205 R945392 0.014 R926745 0.093 R935375 2.8 R945398 0.182 R926753 0.108 R935391 0.223 R945399 0.166 R926757 0.51 R935393 0.45 R945400 17.925 R926763 0.024 R935413 0.195 R945401 0.007 R926780 0.107 R935414 0.152 R945402 0.418 R926782 0.117 R935416 0.196 R945402 0.418 R926791 0.207 R935418 0.558 R945404 9999 R926797 9999 R935431 0.132 R945405 0.168 R926798 9999 R935432 0.05 R945407 9999 R926813 0.405 R935433 0.07 R945412 0.308 R926816 0.062 R935436 0.064 R945413 9999 R926834 0.292 R935437 0.127 R945416 0.515 R926839 0.055 R940233 0.151 R945417 9999 R926891 0.116 R940255 0.771 R945418 9999 R926931 0.255 R940256 3.211 R945419 0.127 R926946 10.218 R940269 0.685 R945422 0.087 R926949 0.076 R940275 0.734 R945423 0.273 R926953 3.05 R940276 0.127 R945424 0.665 R926956 0.38 R940277 0.214 R945426 0.301 R926968 0.235 R940290 0.187 R945427 0.479 R926970 0.057 R940323 0.05 R945432 4444.247 R926971 0.008 R940338 0.028 R945433 0.431 R926975 0.767 R921303 0.003 R945434 9999 R926976 0.421 R940346 0.11 R921302 0.268 R926977 0.007 R940347 0.038 R950349 0.033 R926979 0.013 R940350 0.121 R950367 0.341 R926981 0.01 R940351 0.25 R950368 0.011 R926982 0.028 R940352 0.13 R950373 0.067 R926983 0.012 R940353 0.325 R950428 0.127 R926984 0.459 R940358 0.023 R950430 0.15 R926985 0.203 R940361 0.069 R950431 9999 R926989 0.228 R940363 0.006 R950440 9999 R927016 0.954 R940364 0.001 R950466 1.81 R927017 2.351 R940366 0.003 R950467 9999 R927020 9999 R940367 0.013 R950468 9999 R927042 0.051 R940368 0.001 R950473 19.49 R927048 0.002 R940369 0.043 R950474 9999 R927049 0.004 R940370 0.069 R950475 9999 R927050 0.114 R940371 3.643 R950476 9999 R927051 0.01 R940372 0.253 R940376 0.067 R927056 0.473 R940373 9999 R940380 0.029

These data demonstrate that all of the compounds tested, except for R945142 and R909236 inhibit Syk kinase phosphorylation with IC₅₀s in the submicromolar range. All compounds tested inhibit Syk kinase phosphorylation with IC₅₀s in the micromolar range.

7.18 The Compounds are Effective for the Treatment of Autoimmunity

The in vivo efficacy of certain 2,4-pyrimidinediamine compounds towards autoimmune diseases was evaluated in the reverse passive Arthus reaction, an acute model of antigen-antibody mediated tissue injury, and in several disease models of autoimmunity and inflammation. These models are similar in that antibody to a specific antigen mediates immune complex-triggered (IC-triggered) inflammatory disease and subsequent tissue destruction. IC deposition at specific anatomic sites (central nervous system (CNS) for experimental autoimmune encephalomyelitis (EAE) and synovium for collagen-induced arthritis (CIA)) leads to activation of cells expressing surface FcγR and FcεR, notably mast cells, macrophages, and neutrophils, which results in cytokine release, and neutrophil chemotaxis. Activation of the inflammatory response is responsible for downstream effector responses, including edema, hemorrhage, neutrophil infiltration, and release of pro-inflammatory mediators. The consequences of these IC-triggered events are difficult to identify in autoimmune disorders; nonetheless, many investigators have demonstrated that inhibition of the FcγR signaling pathway in these animal models has resulted in a significant reduction in disease onset and severity.

7.18.1 The Compounds are Effective in Mouse Arthus Reaction

The in vivo efficacy of compounds R921302, R926891, R940323, R940347, and R921303 to inhibit the IC-triggered inflammatory cascade was demonstrated in a mouse model of Reverse Passive Arthus Reaction (RPA reaction).

7.18.1.1 Model

Immune complex (IC)-mediated acute inflammatory tissue injury is implicated in a variety of human autoimmune diseases, including vasculitis syndrome, sick serum syndrome, systemic lupus erythematosus (SLE), rheumatoid arthritis, Goodpasture's syndrome, and glomerulonephritis. The classical experimental model for IC-mediated tissue injury is the reverse passive Arthus reaction. The RPA reaction model is a convenient in vivo method to study localized inflammation, induced by ICs, without systemic effects. Intradermal injection of antibodies (Abs) specific to chicken egg albumin (rabbit anti-OVA IgG), followed by intravenous (IV) injection of antigens (Ags), specifically chicken egg albumin (ovalbumin, OVA), causes perivascular deposition of ICs and a rapid inflammatory response characterized by edema, neutrophil infiltration and hemorrhage at the injection sites. Aspects of the mouse RPA reaction model resemble the inflammatory response of patients with rheumatoid arthritis, SLE and glomerulonephritis.

7.18.1.2 Study Protocol

In this model system, test compounds are administered at several timepoints prior to administration of Abs and Ags. A solution of rabbit anti-OVA IgG (50 μg in 25 μl/mouse) is injected intradermally, and immediately following is an intravenous injection of chicken egg albumin (20 mg/kg of body weight) in a solution containing 1% Evans blue dye. The degree of edema and hemorrhage is measured in the dorsal skin of C57BL/6 mice using the Evan's Blue dye as an indicator of local tissue damage. Purified polyclonal rabbit IgG is used as a control.

Pretreatment time, in which the test compounds are administered prior to Ab/Ag challenge, depends on the pharmacokinetic (PK) properties of each individual compound. Four hours after induction of Arthus reaction, mice are euthanized, and tissues are harvested for assessment of edema. This model system allows us to rapidly screen the in vivo activity of many inhibitors.

7.18.1.3 Results

All compounds tested were administered by the oral route.

R921302, when administered at a dose level of 50 mg/kg, 100 mg/kg, and 200 mg/kg 60 minutes prior to Ab/Ag challenge in C57B16 mice, showed dose-dependent inhibition of edema formation (49.9%, 93.2%, and 99.1%, respectively). Furthermore, R921302 showed not only a prophylactic inhibition of edema, but also therapeutic efficacy in which the edema was inhibited by 77.5% when the compound was administered 30 minutes post-challenge at a dose level of 100 mg/kg.

R940323 and R926891 showed the efficacy of edema inhibition by 32.4% and 54.9%, respectively, when administered at 200 mg/kg, 60 minutes prior to challenge. These compounds are much less bioavailable when administered orally, and systemic exposure levels were approximately 50-fold less that that seen with R921302 (data not shown). R940347 inhibited edema by 89% when administered at a dose level of 100 mg/kg, 2 hours prior to challenge.

Compound R921303 showed 100%, 100%, and 93.6%, inhibition of edema formation when administered at a dose level of 200 mg/kg and a pretreatment time of 30, 60, and 120 minutes, respectively). The compound also demonstrated a dose-dependent inhibition of 65.4%, 81.2% and 100%, at doses of 50 mg/kg, 100 mg/kg and 200 mg/kg, respectively. Results for the compounds tested are summarized in Table 7.

TABLE 7 % inhibition Plasma to vehicle Concentration ± control SEM (ng/ml) Edema Exposure = Compound Dosage Pretreatment Size ± Pretreatment Name (mg/kg) Time (hrs) SEM Time + 4 hours R921302 100 0.5 89.44 ± 4.8  25200 ± 3910 100 1  82.1 ± 10.9 N/A 50 1 50.0 ± 6.4 1149 ± 172 100 1 92.3 ± 4.2 2072 ± 447 200 1 99.1 ± 0.9  4789 ± 1182 R940323 200 0.5  5.5 ± 9.3 2333 ± 618 1  32.4 ± 13.0  878 ± 235 2  26.9 ± 11.2  892 ± 434 R926891 200 0.5 44.8 ± 3.0 163 ± 70 1 46.2 ± 4.1 37.2 ± 8   1.5  28.1 ± 10.6 58.6 ± 19  R921303 200 0.5 100 ± 0  3703 ± 785 1 100 ± 0  2653 ± 833 2 93.3 ± 4.4 2678 ± 496 50 1  64.1 ± 13.3  430 ± 115 100 1 80.5 ± 9.8  983 ± 180 200 1 100 ± 0   2361 ± 1224 R935372 100 0.5 −0.6 ± 6.2 0.6 ± 1  1 23.5 ± 7.4 4.2 ± 4  2  −4.4 ± 17.7 52.65 ± 39   R920410 100 1  42.6 ± 15.1 1216 ± 239 R927050 100 0.5 −0.3 ± 6.6  619 ± 130 1  14.9 ± 20.5  837 ± 104 2 64.0 ± 8.9 557 ± 78 R940350 100 0.5 −15.6 ± 27.2 176 ± 58 1  53.2 ± 15.1 129 ± 55 2  38.9 ± 24.3  96 ± 28 R940347 100 0.5  36.7 ± 22.4 1596 ± 485 1 48.2 ± 5.7 3014 ± 590 2 88.9 ± 9.1 1992 ± 247 R940363 100 0.5 −16.4 ± 10.9  32 ± 10 1  67.6 ± 12.1 42 ± 5 2  52.3 ± 22.7  37 ± 18 R927050 100 1  7 ± 19 1018 ± 189 R927070 50 1  56 ± 15 1755 ± 310 100 1  61 ± 14 2851 ± 712 R940363 100 1 61 ± 8 625 ± 60 R935429 100 1 85 ± 5 401 ± 96 R927070 50 1.5  31.1 ± 17.29 1077 ± 296 100 1.5 55.5 ± 7.7  4095 ± 1187 R935429 50 1.5  −5.1 ± 14.9 164 ± 89 100 1.5  67.1 ± 13.8  206 ± 115 R935429 100 0  −2.8 ± 14.8 NA 100 1 34.08 ± 7.9  NA 100 2 55.5 ± 7.9 NA 100 4  35.0 ± 11.4 NA R927087 50 1.5 −10.4 ± 14.4 26.9 ± 8.0 100 1.5  28.7 ± 16.6  28.7 ± 10.8 R935451 50 1.5 74.9 ± 7.5  385.0 ± 149.4 100 1.5 77.1 ± 8.0 1459.0 ± 444.4 R935451 10 1.5 −14.4 ± 13.3 14.4 ± 1.8 30 1.5 −30.6 ± 15.4  78.0 ± 32.0 R940388 100 1.5 75.0 ± 6.2 44.2 ± 8.9 R921302 50 1 49.9 1.1 100 1 93.2 2.1 200 1 99.1 4.8 R940323 200 1 32.4 0.9 R926891 200 1 54.9 0.04 R940347 100 1 48 nd* 100 2 89 nd R921303 50 1 65.4 0.4 100 1 81.2 0.98 200 1 100 2.4 *nd = not determined

7.18.2 The Compounds are Effective in Mouse Collagen Antibody Induced Arthritis Model

The in vivo efficacy of compound R921302 towards autoimmune diseases was demonstrated a mouse model of collagen antibody-induced arthritis (CAIA).

7.18.2.1 Model

Collagen-induced arthritis (CIA) in rodents is frequently used as one of the experimental models for IC-mediated tissue injury. Administration of type II collagen into mice or rats results in an immune reaction that characteristically involves inflammatory destruction of cartilage and bone of the distal joints with concomitant swelling of surrounding tissues. CIA is commonly used to evaluate compounds that might be of potential use as drugs for treatment of rheumatoid arthritis and other chronic inflammatory conditions.

In recent years, a new technique emerged in CIA modeling, in which the anti-type II collagen antibodies are applied to induce an antibody-mediated CIA. The advantages of the method are: Short time for induction of disease (developing within 24-48 hrs after an intravenous (IV) injection of antibodies); arthritis is inducible in both CIA-susceptible and CIA-resistant mouse strains; and the procedure is ideal for rapid screening of anti-inflammatory therapeutic agents.

Arthrogen-CIA® Arthritis-inducing Monoclonal Antibody Cocktail (Chemicon International Inc.) is administered intravenously to Balb/c mice (2 mg/mouse) on Day 0. Forty-eight hours later, 100 μl of LPS (25 μg) is injected intraperitoneally. On Day 4, toes may appear swollen. By Day 5, one or two paws (particular the hind legs) begin to appear red and swollen. On Day 6, and thereafter, red and swollen paws will remain for at least 1-2 weeks. During the study, the clinical signs of inflammation are scored to evaluate the intensity of edema in the paws. The severity of arthritis is recorded as the sum score of both hind paws for each animal (possible maximum score of 8). The degree of inflammation with involved paws is evaluated by measurement of diameter of the paws. Body weight changes are monitored.

Animals are treated at the time of induction of arthritis, beginning on Day 0. Test compounds and control compounds are administered once a day (q.d.) or twice a day (b.i.d.), via per os (PO), depending on previously established PK profiles.

At the end of the study (1-2 weeks after induction of arthritis), mice are euthanized and the paws are transected at the distal tibia using a guillotine and weighed. The mean±standard error of the mean (SEM) for each group is determined each day from individual animal clinical scores, and hind paw weights for each experimental group are calculated and recorded at study termination. Histopathological evaluation of paws are obtained.

7.18.2.2 Results

Administration of R921302 significantly suppressed the development of arthritis and the severity of the disease (p<0.005), as shown by the changes in mean daily arthritis clinical scores (FIG. 12). The mean daily arthritic scores, from day 4 to 14, in treatment group were reduced between 71 to 92% comparing to that of vehicle control group. The degree of paw inflammation, by measurement of the paw weight, was reduced in animals treated with R921302 compared with the vehicle control group (FIG. 13). At the end of study, the degree of swelling was evaluated by measuring the weight of paws, which is indicated by a 99.9% reduction in group treated with R921302 compared with mean paw weight of the vehicle control group (p<0.002).

Histopathological evaluation of the resected paws revealed a marked synovitis consistent with CIA. Marked lesions were noted in animals treated with saline or vehicle; while lesions of lesser severity were found in R921302 treatment group. The joints were thickened with marked proliferation of the synovium. There is an increase in fibroblasts with a dense infiltration of neutrophils, lymphocytes, monocytes, macrophages and plasma cells. There is vascular proliferation with congestion, hemorrhage and edema. Pannus formation was present in the joint space and there was cartilage destruction. In drug treated group, the joints were close to normal or showed limited inflammation but without cartilage involvement.

TABLE 8 Group Average Histopathological Score (0-15) Treatment Average total score ± SD Saline control 9.8 ± 2.1 Vehicle control 9.3 ± 4.5 R921302 (100 mg/kg), twice 5.1 ± 1.9 daily Naive 0.0 ± 0.0

Arthritic clinical scores and paw edema were reduced by an average of 20% in animals treated with R050 twice daily at a dose level of 100 mg/kg compared with untreated control (vehicle, p=0.1). Paw edema was inhibited by approximately 26% compared with untreated control (vehicle), by measurement of hind paw thickness (p=0.1). R050 did not exhibit arthritis at a dose level of 30 mg/kg.

R070, a salt form of R050, administered at dose levels of 50 or 100 mg/kg twice daily inhibited clinical disease by an average of 39.75% (p<0.0002) or 35.28% (p<0.0004) inhibition, respectively, compared with untreated control (vehicle). Paw thickness was reduced by approximately 50%.

R429, salt of R363, administered twice daily at 50 or 100 mg/kg showed an average of 23.81% (p<0.05) or 20.82% (p=0.05) inhibition of arthritic clinical scores, respectively, compared with untreated control (vehicle). Likewise, paw thickness was reduced.

R347 did not affect arthritic scores at the dose levels tested (30 and 100 mg/kg twice daily).

7.18.3 The Compounds are Effective in Rat Collagen-Induced Arthritis

The in vivo efficacy of compound R921302 towards autoimmune diseases was demonstrated in a rat model of collagen-induced arthritis (CIA).

7.18.3.1 Model Description

Rheumatoid arthritis (RA) is characterized by chronic joint inflammation eventually leading to irreversible cartilage destruction. IgG-containing IC are abundant in the synovial tissue of patients with RA. While it is still debated what role these complexes play in the etiology and pathology of the disease, IC communicate with the hematopoetic cells via the FcγR.

CIA is a widely accepted animal model of RA that results in chronic inflammatory synovitis characterized by pannus formation and joint degradation. In this model, intradermal immunization with native type II collagen, emulsified with incomplete Freund's adjuvant, results in an inflammatory polyarthritis within 10 or 11 days and subsequent joint destruction in 3 to 4 weeks.

7.18.3.2 Study Protocol

Syngeneic LOU rats were immunized with native type II collagen on Day 0, and efficacy of R921302 was evaluated in a prevention regimen and a treatment regimen. In the prevention protocol, either vehicle or various doses of R921302 were administered via oral gavage starting on day of immunization (Day 0). In the treatment protocol, after clinical signs of arthritis developed on Day 10, treatment with R921302 was initiated (300 mg/kg by oral gavage, qd) and continued until sacrifice on Day 28. In both protocols, clinical scores were obtained daily, and body weights are measured twice weekly. At Day 28, radiographic scores were obtained, and serum levels of collagen II antibody were measured by ELISA.

7.18.3.3 Results

By 10 days after immunization, rats developed clinical CIA, as evidenced by an increase in their arthritis scores (FIG. 14). The mean arthritic score gradually increased in the rats treated with vehicle alone after Day 10, and by Day 28 the mean clinical score reached 6.75±0.57. Mean clinical scores in animals treated from the day of immunization (Day 0) with the high dose of R921302 (300 mg/kg/day) were significantly reduced (p<0.01) on Days 10-28 compared with vehicle controls. In the rats treated with 300 mg/kg R921302 at disease onset, there was a significantly lower arthritis score beginning on Day 16, and this difference was observed until the end of the study on Day 28. Blinded radiographic scores (scale 0-6) obtained on Day 28 of CIA were 4.8±0.056 in the vehicle group compared with 2.5±0.0.16, 2.4±0.006, and 0.13±0.000001 in animals treated once daily with 75, 150, and 300 mg/kg/day, respectively, in a prevention regimen, and 0.45±0.031 in animals treated once daily with 300 mg/kg/day at onset of disease. R921302 treatment at 300 mg/kg/day, either prophylactically (at immunization) or after disease onset precluded the development of erosions and reduced soft tissue swelling. Similarly, R921302 treatment resulted in marked reduction of serum anti-collagen II antibody (data not shown).

7.18.4 The Compounds are Effective in Mouse Experimental Autoimmune Encephalomyelitis

The in vivo efficacy of compound R921302 towards autoimmune diseases was demonstrated in a mouse model of experimental autoimmune encephalomyelitis (EAE)

7.18.4.1 Model Description

EAE is a useful model for multiple sclerosis (MS), an autoimmune disease of the CNS that is caused by immune-cell infiltration of the CNS white matter. Inflammation and subsequent destruction of myelin cause progressive paralysis. Like the human disease, EAE is associated with peripheral activation of T cells autoreactive with myelin proteins, such as myelin basic protein (MBP), proteolipid protein (PLP), or myelin oligodendrocyte protein (MOG). Activated neuroantigen-specific T cells pass the blood-brain barrier, leading to focal mononuclear cell infiltration and demyelination. EAE can be induced in susceptible mouse strains by immunization with myelin-specific proteins in combination with adjuvant. In the SJL mouse model used in these studies, hind limb and tail paralysis is apparent by Day 10 after immunization, the peak of disease severity is observed between Days 10 and 14, and a cycle of partial spontaneous remission followed by relapse can be observed up to Day 35. The results described below demonstrate the potential of the test agent (R921302) to suppress disease severity and prevent relapse of disease symptoms that may be the result of FcγR-mediated cytokine release from immune cells.

7.18.4.2 Study Protocol

In the SJL murine model of EAE, each mouse is sensitized with PLP/CFA. (150 μg PLP139-151 with 200 pg CFA in 0.05 ml of homogenate on four sites of hind flank for a total of 0.2 ml emulsion is used to induce EAE). In a suppression protocol, either vehicle or various doses of R921302 are administered via oral gavage starting on the day of immunization (Day 0). In a treatment protocol, at onset of disease, animals are separated to achieve groups with a similar mean clinical score at onset and administered vehicle or various dose frequencies of test articles via oral gavage. In both protocols, clinical scores are monitored daily, and body weights are measured twice weekly.

7.18.4.3 Results

By 10 days after PLP immunization, SJL mice developed clinical EAE, as evidenced by an increase in their mean clinical scores (FIG. 15). The paralytic score gradually increased in the animals treated with vehicle only from the day of immunization (Day 0), and by Day 14 the mean score reached a peak of 5.1+0.3. At disease peak (Day 14), the mean clinical score in animals treated with either 100 mg/kg daily or 100 mg/kg twice daily was significantly reduced (p<0.05, 4.3+1.3 and 4.3+1.4, respectively). By Day 16, all animals exhibited a partial remission of mean clinical severity, which is a characteristic of the SJL model. The markedly lower clinical scores in animals treated twice daily with 100 mg/kg R921302 remained significant (p<0.05) throughout the experiment until the animals were sacrificed on Day 30. These lower scores throughout the treatment period are reflected in the significantly lower cumulative disease index (CDI) and increase in cumulative weight index (CWI) as seen in Table 9. In the group treated with vehicle only, 2/5 of the mice relapsed. In the 100 mg/kg/day group, 3/8 of the mice relapsed. None of the mice in the 100 mg/kg twice daily group relapsed.

TABLE 9 SJL female mice treated with Rigel compound R921302 starting on day of immunization with 150 μg PLP 139-151/200 μg MTB (CFA) Incidence Onset Peak Mortality CDI CWI Placebo 10/10 11.8 ± 0.5 5.1 ± 0.3 1/10^(a) 53.2 ± 7.1 118.1 ± 6.4 Control 100 mg/kg 10/10 12.3 ± 0.7 4.3 ± 1.3 0/10 44.1 ± 14.5 124.4 ± 6.0 1x/day 100 mg/kg 10/10 13.0 ± 1.2^(b) 4.3 ± 1.4 3/10^(a) 33.7 ± 11.4^(b) 133.5 ± 6.8^(b) 2x/day CDI = Cumulative Disease Index to day +26 CWI = Cumulative Weight Index to day +23 ^(a)= Mortality due to non-EAE, feeding related injuries or sacrificed hydrocephalic animals. ^(b)= Significant difference between Control vs. Experimental groups (p < 0.05) determined via Students two-tailed t test.

SJL mice treated with R921302 at disease onset (Day 11) at a dose level of 200 mg/kg twice daily showed a significant decrease (p=0.003) in CDI (53.5±16.9 in animals treated with R921302 compared with 72.9±8.9 in the animals treated with vehicle alone). Further, there was a dramatic decrease in the number of relapses in animals treated with R921302 (2/12) compared with the number of relapses in animals treated with vehicle (7/11). Results are summarized in Table 10 and FIG. 16.

TABLE 10 SJL female mice treated with Rigel compound R921302 starting on day of onset Mean Inci- score at Mor- dence treatment Peak tality Relapses CDI Control 11/11 3.9 ± 1.6 5.0 ± 0.4 0/11 7/11 72.9 ± 8.9  200 12/12 3.4 ± 1.6 4.3 ± 0.7 1/12 2/12 53.5 ± 16.9 mg/kg 2x/day P value 1.00 0.48 0.02 0.97 0.055 0.003 CDI = Cumulative Disease Index to day +27

7.18.5 The 2,4-Pyrimidinediamine Compounds of the Invention Inhibit T-Cell Activation 7.18.5.1 Description

The ability of the 2,4-pyrimidinediamine compounds of the invention to inhibit activation of T-Cells was shown using a variety of assays utilizing a Jurkat T-cell cell line and Primary T-cell cultures Inhibition of activation of Jurkat T-cells in response to T-cell receptor (TCR) stimulation was measured by quantifying the upregulation of the cell surface marker CD69. Inhibition of primary T-cell activation was measured by quantifying the release of cytokines, including tumor necrosis factor alpha (TNF), interleukin 2 (IL-2), interleukin 4 (IL-4) interferon gamma (IFNg) and granulocyte macrophage colony stimulating factor (GMSCF), in response to TCR/CD28 co-stimulation.

7.18.5.2 Screening for Inhibition of Jurkat T-Cell Activation

Human Jurkat T-cells (clone N) were routinely cultured in RPMI 1640 medium (Mediatech) supplemented with 10% fetal calf serum (FBS) (Hyclone), penicillin and streptamycin. The screening process took place over three days.

On the first day of the screen, cultured cells were spun down on a centrifuge (1000 rpm, 5 minutes) and resuspended at 3.0×10⁵ cells/ml in RPMI+5% FBS. On the second day of the screen, cells were spun down at 1000 rpm for 5 minutes and resuspended in RPMI+5% FBS at 1.3×10⁵ cells/ml. 85 μl of this cell suspension were added to the wells of U-bottom 96 well plates (Corning). 85 μl of compound or diluted RPMI+5% FBS (as a control) only was added to each well and incubated at 37° C. for 1 hour. The cells were then stimulated with anti-TCR(C305) at: 500 ng/ml by adding a 8× solution in 25 μl to the plated cells. The cells were then incubated at 37° C. for 20 hrs.

On the third day of the screen, the plates were spun at 2500 RPM for 1 minute on a Beckman GS-6R centrifuge, and the medium was then removed. 50 μl staining solution (1:100 dilution of anti-CD69-APC antibody (Becton Dickenson) in PBS+2% FBS) was then added to each well, followed by incubation of the plates 4 degrees for 20 minutes in the dark. 150 μl of wash buffer (PBS+2% FBS) was then added to each well, and the plates were spun at 3000 RPM for 1 minute. The supernatant wase again removed, and the pellet was resuspended by vortexing gently. 75 μl of PBS+2% FBS+Cytofix (1:4 dilution) was then added, the plates gently vortexed and wrap in aluminum foil. Cells from the plates were read using a flow cytometer coupled to an automated liquid handling system.

Varied concentrations of compound were compared to solvent only to determine the inhibition of T-cell activation IC₅₀ of each compound. Representative IC₅₀s for 2,4-pyrimidinediamine compounds of the invention are shown in Table 11.

7.18.5.3 Isolation of Primary T-Cells

2E8-4E8 PBMC or proliferating T cells grown in rIL-2 from healthy human donors were suspended in PBS were spun down (1500 rpm, 8-10 minutes) and resuspended in 100 ml RPMI Complete media (1% Pen-Strep, 1% L-Glutamine, 10 mM HEPES). The cells were plated in T175 flasks (37° C., 5% CO₂) and monocytes were allowed to adhere for 2-3 hours. After monocyte attachment, non-adherent cells were harvested, counted by hemocytometer, washed several times with PBS then resuspended in Yssels Complete Media (Modified IMDM Media with 1% Human AB Serum, 1% Pen-Strep, 1% L-Glutamine, 10 mM HEPES) at 1.5 4E6 cells/mL. 90 uL of the cell dilution were then added to compounds diluted to 2× in Yssel's media and incubated for 30 minutes at 37° C. (5% CO₂). After this preincubation step the compound/cell mixture was transferred to stimulation plates, as described below.

7.18.5.4 Screening for Inhibition of Cytokine Production in Stimulated Primary T-Cell

Stimulation plates were prepared by coating 96 well plates with 5 μg/ml αCD3 (BD PharMingen, Catalog#555336)+10 μg/ml αCD28 (Beckman Coulter, Catalog# IM1376) in PBS (no Ca²⁺/Mg²⁺) at 37° C. (5% CO₂) for at 3-5 hours. After incubation with the stimulation antibodies, the cocktail was removed and the plates washed 3 times with PBS prior to addition of the primary T cell/compound mixture.

The compound/cell mixture was transferred to the stimulation plates and incubated for 18 hr at 37° C. (5% CO₂). After the cell stimulation, ˜150 μl supernatant were transferred from each well into 96-well filter plates (Corning PVDF Filter Plates) spun down (2000 rpm, 2-3 minutes) and either used immediately for ELISA or LUMINEX measurements or frozen down at −80° C. for future use.

IL-2 ELISAs were performed using the Quantikine Human IL-2 ELISA kit (R&D Systems, Catalog# D2050) as described by the manufacturer and absorption was measured on a spectrophotometer at 450 nm wavelength. Blank values were substracted and absorbances were converted to pg/mL based on the standard curve.

Luminex immunoassay multiplexing for TNF, IL-2, GMSCF, IL-4 and IFNg was performed essentially as described by the manufacturer (Upstate Biotechnology). Essentially 50 uL of sample was diluted into 50 uL assay diluent and 50 uL incubation buffer, then incubated with 100 uL of the diluted detection antibody for 1 hr at RT in the dark. The filter plate was washed 2× in Wash Buffer, then incubated with 100 uL of the diluted secondary reagent (SAV-RPE) for 30 min at RT in the dark. Finally the plates were washed 3 times and bead identification and RPE fluorescent measured by the Luminex instrument.

Varied concentrations of compound were compared to solvent only to determine the inhibition of T-cell activation IC₅₀ of each compound. Representative IC₅₀s for 2,4-pyrimidinediamine compounds of the invention are shown in Table 11.

7.18.6 The 2,4-Pyrimidinediamine Compounds of the Invention Inhibit B-Cell Activation 7.18.6.1 Description

The ability of the 2,4-pyrimidinediamine compounds of the invention to inhibit activation of B-cells was shown using primary B-cells in a cell surface marker assay using a fluorescence activated cell sorter (FACS). Inhibition of activation of primary B-cells in response to B-cell receptor (BCR) stimulation was measured by quantifying the upregulation of the cell surface marker CD69.

7.18.6.2 Isolation of Primary B-Cells

Primary human B-cells were isolated from buffy coat, the white cell layer that forms between the red cells and the platelets when anti-coagulated blood is centrifuged, or from fresh blood using CD19-Dynal® beads and a FACS. Buffy coat was obtained from the Stanford Medical School Blood Centre, prepared on the same day by the blood bank, stored and transported cold (with ice). The buffy coat (approx 35 mL) was placed in a 500 mL conical sterile centrifuge pot and cooled on ice, then diluted with cold PBS containing 0.2% BSA (Sigma: A7638) and sodium citrate (0.1%, Sigma: S-5570) (P-B-C) to a total volume of 200 mL and mixed gently. Fresh blood was collected from donors in 10 mL vacutainers containing heparin (1 vacutainer collects approximately 8.5 mL blood). The blood was cooled on ice, transferred into 50 mL falcon tubes (20 mL/tube) or a 500 mL conical sterile centrifuge pot, and diluted with an equal volume P-B-C.

25 mL diluted blood or buffy coat was layered onto 15 mL cold ficoll and placed back on ice. The ficoll layered blood was centrifuged (Beckman GS-6R) for 45 minutes at 2000 rpm, 4° C. to separate the Peripheral Blood Mononuclear Cells (PBMC) from the Red Blood Cells (RBC) and granulocytes. The top aqueous layer was then aspirated until 1 inch above the PBMC layer. The PBMCs were transferred from every 2 ficoll tubes into one clean 50 mL falcon tube (=approx 10 mL/tube). The transferred PBMCs were diluted 5× with icecold PBS with 0.2% BSA (P-B) and centrifuged for 20 min at 1400 rpm and 4° C. The supernatant (this may be cloudy) was then aspirated and the PBMCs resuspended into 25 mL P-B and the cells counted (using a 1:5 dilution) and kept on ice.

The cells were then positively selected using anti-CD19 antibody coupled to magnetic beads (Dynal®) as per manufacturer's instructions. The approximate required amount of CD19-Dynal® beads (CD19-coated dyna beads M-450 (pabB), Dynal®) was calculated by estimating the number of B-cells as 5% of PBMCs counted and adding approximately 10 beads per cell from the bead stock (4×10⁸ beads/mL). The CD19-Dynal® beads were washed 2× in P-B in a 5 mL tube using the Dynal® magnet, then added into the suspended PBMCs. This mixture was then passed through the Dynal® magnet and washed several times to separate the bead-bound cells.

7.18.6.3 Screening Compounds for Inhibition of B-Cell Activation

After separation, the beads and antibody were removed using Dynal® CD19-DETACHaBEAD® for 45 min at 30° C. Yield is typically 2×10⁷ B-cells per buffy coat. B-cells were washed and resuspended as 1E6 cells/mL in RPMI1640+10% FBS+Penicillin/Streptavidin+1 ng/mL IFNα8. Cells were rested overnight at 37° C. and 5% CO₂.

The following day, cells were washed and resuspended in RPMI+2.5% FBS to 1×10⁶ cells/mL. Cells were then aliquoted into a V-bottom 96-well plate (Corning) at 65 uL cells per well. By robot, 65 uL of a 2× compound was added to the cells with final concentration of DMSO at 0.2%, and incubated for 1 hr at 37° C. Cells were then stimulated with 20 uL 7.5× α-IgM from Jackson laboratories (final 5 ug/mL) for 24 hrs. At day 3, the cells were spun down and stained for CD69 and analyzed by FACS gated on the live cells (by light scatter).

Varied concentrations of compound were compared to solvent only to determine the inhibition of B-cell activation IC₅₀ of each compound. Representative IC₅₀s for 2,4-pyrimidinediamine compounds of the invention are shown in Table 11.

7.18.7 The 2,4-Pyrimidinediamine Compounds of the Invention Inhibit Macrophage Activation 7.18.7.1 Description

The ability of the 2,4-pyrimidinediamine compounds of the invention to inhibit activation of differentiated macrophages was shown by measuring the release of cytokines from stimulated macrophages. Release of tumor necrosis factor alpha (TNF) and interleukin 6 (IL-6) was quantified in response to IgG or LPS stimulation.

7.18.7.2 Purification and Culture of Human Macrophages

CD14+ monocytes were purified from PBMC (Allcells # PB002) using the Monocyte Isolation kit (Miltenyi biotec #130-045-501) as per the manufacturer's instructions. Purity was assessed by measuring the percentage of CD14+ cells by flow cytometry. Typically >90% purity is achieved. The purified CD14+ cells are then plated out (6×10⁶·cells/150 cm TC dish in 15 mls media) in Macrophage-SFM (Gibco #12065-074) with 100 ng/ml of M-CSF (Pepro Tech #300-25) and allowed to differentiate for five days. At the end of that period, cell morphology and cell surface markers (CD14, HLA-DR, B7.1, B7.2, CD64, CD32, and CD16) reflected the presence of mature differentiated macrophage.

7.18.7.3 Stimulation with IgG

Immulon 4HBX 96 well plates (VWR #62402-959) were coated with pooled human IgG (Jackson Immunoresearch lab#009-000-003) at 10 ug/well overnight at 4° C. or 1 hr at 37° C. A negative control consisting of the F(ab′)2 fragment was also coated to assess background stimulation. Unbound antibody was washed away 2× with 200 ul PBS. 20 ul of 5× compound was added to each well, followed by the addition 15 k cells of differentiated macrophage in 80 uL that had been scraped off of the plates. The cells were incubated for 16 hr in a 37° C. incubator, and supernatants were collected for Luminex analysis for IL-6 and TNFα, essentially as described for the primary T-cells, above.

7.18.7.4 Stimulation with LPS

For stimulation with LPS, 10 uL of a 10× stock solution was added to the preincubated cell-compound mixture to a final concentration of 10 ng/mL. The cells were then incubated for 16 hr at 37° C. and supernatants were analyzed as described above.

Varied concentrations of compound were compared to solvent only to determine the IC₅₀ of each compound for each cytokine. Representative IC₅₀s for 2,4-pyrimidinediamine compounds of the invention are shown in Table 11.

TABLE 11 Monocytes/ Jurkat 1° T-Cell 1° B-Cell Macrophage CD69 IC50 TNF IC50 IL2 IC50 GMSCF IC50 IL4 IC50 IFNg CD69 IC50 TNF IC50 IL-6 IC50 Compound (in μM) (in μM) (in μM) (in μM) (in μM) IC50 (in μM) (in μM) (in μM) (in μM) R070790 9999 R908696 9999 R908697 9999 R908698 3.748 R908699 1.033 R908700 13.724 R908701 0.302 R908702 0.37 R908703 1.399 R908704 3.037 R908705 5.876 R908706 0.405 R908707 9.372 R908709 3.394 R908710 4.277 R908711 4.564 R908712 0.348 R908734 3.555 R908953 1.982 R909236 9999 R909237 9999 R909238 5.021 R909239 3.063 R909240 2.845 R909241 3.52 R909242 3.8 R909243 2.245 R921219 0.441 0.546 0.131 R909245 0.78 R909246 2.166 R909247 3 R909248 33.258 R909249 9999 R909250 9999 R909251 0.664 R909252 0.655 R909253 3.082 R909255 1.973 R909259 9999 R909260 3.329 R909261 2.935 R909263 6.195 R909264 3.241 R909265 11.988 R909266 12.983 R909267 9999 R909268 0.997 R909290 1.562 R909292 3.315 R909317 0.224 0.595 1.324 1.743 0.876 1.573 R909322 3.028 1.259 0.839 R920395 0.726 R920410 1.981 2.989 3.36 3.2 0.546 4.307 0.706 R920664 9999 R920665 10.883 R920666 9999 R920668 9999 R920669 19.813 R920670 14.322 R920671 9999 R920672 9999 R920818 9999 R920819 9999 R920820 9999 R920846 10.404 R920860 9999 R920861 3.28 R920893 1.4 R920894 2.024 R920910 2.38 R920917 2.649 R925734 9999 R925745 9999 R925746 9999 R925747 9999 R925755 1.906 R925757 9999 R925758 18.209 R925760 20.246 R925765 9999 R925766 9999 R925767 9999 R925768 9999 R925769 9999 R925770 9999 R925771 7.187 R925772 9999 R925773 14.414 R925774 7.498 R925775 9999 R925776 17.059 R925778 3.398 R925779 9999 R925783 9999 R925784 9999 R925785 3.117 R925786 9999 R925787 9999 R925788 16.898 R925790 16.992 R925791 9999 R925792 8.65 R925794 9999 R925795 9999 R925796 1.827 R925797 1.511 R925798 9999 R925799 9999 R925800 9999 R925801 9999 R925802 9999 R925803 9999 R925804 9999 R925805 9999 R925806 9999 R925807 9999 R925808 9999 R925810 21.332 R925811 9999 R925812 9999 R925814 14.163 R925815 9999 R925816 4.664 R925819 9999 R925820 9999 R925821 9999 R925822 9999 R925823 9.326 R925838 9999 R925842 9999 R925845 6.968 R925846 9999 R925849 8.022 R925852 9999 R925853 9999 R925855 9999 R925856 9999 R925857 9999 R925858 9999 R925860 41.865 R925861 20.195 R925862 9999 R925863 2.962 R925864 19.127 R925865 9999 R926016 9999 R926017 20.775 R926018 9999 R926037 9999 R926038 9999 R926039 9999 R926058 9999 R926064 9999 R926065 6.731 R926068 11.416 R926069 4.307 R926072 9999 R926086 6.635 R926108 10.373 R926109 16.117 R926110 3.474 R921218 3.935 3.24 1.081 R926113 4.379 R926114 9.913 R926145 17.689 R926146 9999 R926147 9999 R926206 9999 R926209 9999 R926210 4.379 R926211 14.957 R926212 0.56 R926213 8.864 44 R926218 9999 R926220 9999 R926221 9999 R926222 9999 R926223 9999 R926224 9999 R926225 9999 R926228 9999 R926229 9999 R926230 9999 R926234 9999 R926237 9999 R926238 9999 R926240 9999 R926241 13.768 R926242 3.824 R926243 2.986 R926245 11.086 R926248 1.537 R926249 0.954 R926252 9999 R926253 9999 R926254 9999 R926255 9999 R926256 9999 R926257 9999 R926258 9999 R926259 12.96 R926319 15.584 R926320 9999 R926321 1.293 R926325 9999 R926331 9999 R926339 2.149 R926340 9999 R926341 3.676 R926376 9999 R926386 9999 R926387 3.852 R926394 9999 R926395 17.741 R926396 6.594 R926397 12.469 R926398 9999 R926399 9999 R926400 9999 R926401 9999 R926402 9999 R926403 9999 R926404 9999 R926405 7.617 R926408 9999 R926409 3.539 R926411 16.926 R926412 2.383 R926461 3.388 R926467 9999 R926469 9999 R926474 10.775 R926475 9999 R926476 3.904 R926477 9999 R926479 9999 R926480 9999 R926481 9999 R926482 8.261 R926483 9999 R926484 9999 R926485 9999 R926486 1.745 R926487 48.937 R926488 2.429 R926489 9999 R926491 2.727 R926492 3.335 R926493 3.524 R926494 12.507 R926495 11.904 0.643 R926496 4.387 R926497 3.267 R926498 5.732 R926499 0.56 R926500 2.367 R926501 1.681 R926502 1.626 R926503 2.599 R926504 1.784 R926505 1.145 R926506 2.676 R926508 1.006 0.917 0.948 R926509 1.078 R926510 0.122 R926511 1.729 R926514 15.6 R926516 17.782 R926526 9999 R926527 21.197 R926528 9999 R926535 9999 R926536 9999 R926555 9999 R926559 11.248 R926560 9999 R926561 9999 R926562 1.246 R926563 9999 R926564 9999 R926565 9999 R926566 9999 R926567 9999 R926569 9999 R926571 9999 R926572 9999 R926574 9999 R926576 9999 R926585 9999 R926586 9999 R926587 9999 R926588 9999 R926589 9999 R926591 9999 R926593 1.282 R926594 1.252 R926595 9999 R926604 9999 R926605 9999 R926614 6.537 R926615 1.871 R926616 1.912 R926617 9999 R926620 9999 R926623 10.015 R926662 9999 R926675 2.369 R926676 9999 R926680 5.703 R926681 2.002 R926682 5.946 R926683 7.635 R926688 3.779 R926690 13.398 R926696 7.645 R926698 9999 R926699 1.861 R926700 0.51 R926701 9999 R926702 18.583 R926703 7.873 R926704 9.271 R926705 2.651 R926706 9999 R926707 2.683 R926708 3.299 R926709 2.47 R926710 4.273 R926711 3.788 R926712 6.351 R926713 8.219 R926714 5.632 R926715 2.357 R926716 3.618 R926717 3.75 R926718 12.441 R926719 9999 R926720 9999 R926721 3.461 R926722 9999 R926723 9999 R926724 9999 R926725 3.368 R926726 9999 R926727 9999 R926728 9999 R926730 1.84 R926731 9999 R926732 5.256 R926733 3.594 R926734 11.276 R926735 5.982 R926736 14.12 R926737 2.384 R926738 2.216 R926739 2.093 R926740 9999 R926741 4.593 R926742 0.717 R926743 9999 R926744 9999 R926745 1.484 1.498 R926746 3.696 R926747 3.278 R926748 2.769 R926749 4.684 R926750 0.535 R926751 5.592 R926752 1.734 R926753 0.393 R926754 13.245 R926755 7.364 R926756 3.774 R926757 2.737 R926759 1.71 R926760 10.25 R926761 0.694 R926762 0.703 R926763 3.717 R926764 2.165 R926765 8.003 R926766 4.24 R926767 2.667 R926768 0.973 R926769 2.79 R926770 0.891 R926771 3.473 R926772 2.043 R926773 1.844 R926774 12.741 R926775 9999 R926776 12.475 R926777 9999 R926778 9999 R926779 9999 R926780 2.158 R926781 9.811 R926782 1.221 R926783 2.95 R926784 2.379 R926785 2.583 R926786 7.361 R926787 9999 R926788 9999 R926789 9999 R926790 9999 R926791 1.751 R926792 9.975 R926795 9999 R926796 4.205 R926797 9999 R926798 9999 R926799 9999 R926800 9999 R926801 9999 R926802 5.909 R926803 9999 R926804 9999 R926805 9999 R926806 6.076 R926807 10.136 R926808 1.76 R926809 9999 R926810 5.069 R926811 1.284 R926812 6.76 R926813 5.101 R926814 9999 R926815 9999 R926816 0.739 R926826 3.732 R926827 2.135 R926828 1.006 R926829 3.095 R926830 4.161 R926831 1.271 R926832 2.988 R926833 11.797 R926834 2.568 R926835 3.585 R926836 14.528 R926837 9999 R926838 10.684 R926839 2.485 R926840 12.234 R926841 3.279 R926842 9999 R926843 9999 R926844 9999 R926845 9999 R926846 9999 R926847 11.782 R926848 1.72 R926851 3.089 R926852 9999 R926853 9999 R926854 48.759 R926855 9999 R926856 9999 R926857 9999 R926858 9999 R926859 9999 R926860 9999 R926861 9999 R926862 7.746 R926863 9999 R926866 9999 R926869 9999 R926873 9999 R926875 9999 R926876 9999 R926877 9999 R926878 9999 R926879 2.554 R926880 6.239 R926881 11.025 R926882 9.049 R926883 9999 R926884 9999 R926885 9999 R926886 1.136 R926887 5.92 R926888 5.582 R926889 9999 R926890 11.291 R926891 1.548 0.803 1.135 0.942 R926892 1.635 R926893 9999 R926894 9999 R926895 9999 R926896 9999 R926897 9999 R926898 9999 R926899 9999 R926900 9999 R926902 9999 R926903 9999 R926904 1.363 R926905 6.488 R926906 9999 R926907 17.14 R926908 30.57 R926909 4.65 R926910 9999 R926911 9999 R926912 9999 R926913 5.652 R926914 9999 R926915 9999 R926917 4.741 R926918 4.689 R926919 9999 R926920 9999 R926921 9999 R926922 6.123 R926923 7.203 R926924 3.228 R926925 5.868 R926926 13.105 R926927 5.527 R926928 9999 R926929 3.998 R926930 10.481 R926931 2.933 R926932 2.907 R926933 2.79 R926934 6.011 R926935 11.794 R926936 7.883 R926937 9999 R926938 9999 R926939 9999 R926940 9999 R926941 9999 R926942 9999 R926943 18.527 R926944 3.43 R926945 4.243 R926946 9.4 R926947 13.298 R926956 0.749 R926968 2.024 R926976 1.16 4.369 7.618 R926982 0.394 R927016 7.156 R927017 8.157 R927018 17.68 R927019 9999 R927050 0.112 0.6 0.928 1.118 0.275 0.916 0.438 0.108 0.066 R927064 2.735 9999 9999 9999 1.754 R927069 0.93 8.505 5.65 R935000 9999 R935001 9999 R935002 9999 R935003 9999 R935004 9999 R935005 9999 R935006 9999 R935016 5.363 R935019 9999 R935020 9999 R935021 9999 R935023 9999 R935025 7.949 R935075 5.366 R935076 9999 R935077 9999 R935114 9999 R935117 9999 R935134 9999 36.11 R935135 9999 R935136 9999 R935137 24.124 R935138 0.46 R935139 10.963 R935140 2.158 R935141 9999 R935142 9.665 R935143 3.843 R935144 9999 13.31 R935145 5.339 R935146 9999 R935147 1.981 R935148 9999 R935149 9999 R935150 20.372 R935151 1.961 R935152 19.866 R935153 7.071 R935154 1.646 R935155 9999 R935156 1.845 R935157 9999 R935158 2.47 R935159 9999 R935160 2.37 R935161 3.134 R935162 3.377 R935163 9999 R935164 3.319 R935165 9999 R935166 9999 R935167 9999 R935168 3.71 R935169 7.539 R935170 6.027 R935171 3.927 R935172 9999 R935173 3.908 R935174 3.99 R935175 1.743 R935176 1.981 R935177 4.154 R935178 3.04 R935179 2.999 R935180 3.571 R935181 8.983 R935182 23.856 R935183 2.271 R935184 4.082 R935185 4.107 R935186 1.095 R935187 9999 R935188 1.803 R935189 0.736 R935190 3.472 R935191 2.938 R935192 5.39 R935193 1.596 R935194 0.732 R935196 1.103 R935197 2.428 R935198 1.453 R935199 2.509 R935202 1.941 R935203 9999 R935204 3.869 R935205 10.715 R935206 9999 R935207 9999 R935208 2.877 R935209 9999 R935211 7.06 R935212 4.682 R935213 3.089 R935214 1.378 R935215 7.955 R935216 3.475 R935217 9999 R935218 22.692 R935219 5.567 R935220 8.067 R935221 9999 R935222 3.535 R935223 4.574 R935224 9999 R935225 7.422 R935237 9999 R935238 6.727 R935239 1.726 R935240 2.709 R935242 9999 R935248 1.898 R935249 4.833 R935250 6.236 R935255 0.668 R935256 0.92 R935258 6.26 R935259 3.458 R935261 2.181 R935262 3.113 R935263 2.017 R935264 1.408 R935266 9999 R935267 3.93 R935268 2.906 R935269 7.578 R935271 0.858 R935279 1.984 R935286 2.497 R935287 1.697 R935288 9999 R935289 5.338 R935290 3.43 R935291 3.139 R935292 3.61 R935293 1.337 R935294 8.16 R935295 14.241 R935296 9999 R935297 5.701 R935298 2.317 R935299 0.824 R935300 3.384 R935301 2.317 R935302 0.8 R935303 0.653 R935304 0.497 R935305 1.834 R935306 4.726 R935307 1.407 R935308 1.265 R935309 0.779 R935310 0.88 R935320 9999 R935321 9999 R935322 9999 R935323 9999 R935324 9999 R935336 2.878 R935337 2.537 R935338 5.891 R935339 9999 R935340 9999 R935366 4.182 R935368 9999 R935372 30.713 R935391 6.041 0.669 1.157 0.959 R935393 9999 R940079 9999 R940089 9999 R940090 9999 R940095 9999 R940100 9999 R940110 9999 R940215 9999 R940216 1.283 R940217 9999 R940222 9.471 R940233 2.171 R940253 17.367 R940254 3.763 R940255 1.509 R940256 4.745 R940257 9999 R940258 9999 R940260 9999 R940261 10.948 R940262 6.448 R940263 10.05 R940264 9999 R940265 5.563 R940266 9999 R940267 9999 R940269 1.895 R940270 9999 R940271 9999 R940275 16.37 R940276 2.532 R940277 1.223 R940280 9999 R940281 9999 R940282 6.709 R940283 9999 R940284 78.15 R940285 9999 R940286 4.4 R940287 6.197 R940288 3.485 R940289 3.646 R940290 1.16 R940291 9.446 R940292 2.781 R940293 9999 R940294 9999 R940296 1.23 R940297 9999 R940299 24.942 R940300 9.284 R940301 1.314 R940304 9999 R940306 11.036 R940307 2.063 R940309 9999 R940311 4.123 R940312 16.178 R940314 7.032 R940316 4.278 R940317 3.282 R940318 1.387 R940320 7.818 R940321 3.68 R940322 4.57 R940323 0.557 0.11 R940336 9999 R940337 1.821 R940338 0.708 R940342 5.124 R921303 0.423 0.796 1.02 1.178 0.366 1.28 0.217 R940344 7.735 R940345 5.395 R940346 2.086 R940347 0.581 0.0992 1.894 1.613 0.212 1.673 0.47 0.038 0.019 R940350 0.308 1.513 2.993 2.45 0.501 2.471 0.297 R940352 3.53 0.876 R940353 20.699 R940358 0.159 R940361 0.39 R940363 0.141 0.242 0.133 0.095 R940366 0.086 0.086 0.097 R945025 7.033 R945032 15.179 R945033 9999 R945034 9999 R945035 9999 R945036 9999 R945037 9999 R945038 9999 R945040 9999 R945041 9999 R945042 9999 R945043 9999 R945045 7.602 R945046 4.078 R945047 3.206 R945048 2.231 R945051 9999 R945052 9999 R945053 2.674 R945056 9999 R945057 9999 R945060 6.076 R945061 9999 R945062 9999 R945063 6.038 R945064 4.684 R945065 14.427 R945066 43.243 R945067 9999 R945068 9999 R945070 9999 R945071 0.631 R945096 2.802 R945097 9999 R945109 9.637 R945110 9999 R945117 9999 R945118 9.492 R945124 6.161 R945125 9999 R945126 9999 R945127 11.084 R945128 4.311 R945129 6.08 R945130 9999 R945131 19.162 R945132 20.194 R945133 9.14 R945135 4.367 R945137 5.429 R945138 9999 R945139 13.869 R945140 2.094 R945142 1.88 R945144 1.656 R945145 9999 R945146 9999 R945147 9999 R945148 16.217 R945149 1.226 R945150 1.112 R945151 9999 R945152 9999 R945153 9.738 R945155 7.067 R945156 2.29 R945157 1.477 R945162 9999 R945163 9999 R945164 9999 R945165 9999 R945166 9999 R945167 5.072 R945168 9999 R945169 2.38 R945170 4.123 R945171 3.194 R945172 3.132 R945173 2.884 R945175 3.787 R945236 2.921 R945237 0.838 R945242 1.707 R945263 4.467 R921304 0.141 1.497 2.772 1.567 0.366 2.894 0.167 R945298 9.467 R945299 1.063 R950083 9999 R950090 9999 R921302 3.513 1.628 5.185 3.207 0.245 3.896 1.17 R950092 9999 R950093 11.28 R950100 5.67 R950107 5.424 R950108 9999 R950109 12.782 R950120 12.062 R950121 6.265 R950122 13.894 R950123 9999 R950125 9999 R950129 6.88 R950130 9999 R950131 9999 R950132 4.638 R950133 4.701 R950134 6.455 R950135 9999 R950137 5.904 R950138 9999 R950139 5.454 R950140 22.366 R950141 2.376 R950142 29.078 R950143 4.569 R950144 9999 R950145 6.13 R950146 9999 R950147 14.803 R950148 9999 R950149 9999 R950150 9999 R950151 14.221 R950152 2.654 R950153 9999 R950154 9999 R950155 9999 R950156 9999 R950157 9999 R950158 21.381 R950159 8.446 R950160 9999 R950162 8.918 R950163 24.106 R950164 18.213 R950165 7.594 R950166 9999 R950167 9999 R950168 10.692 R950169 9999 R950170 9999 R950171 4.358 R950172 23.117 R950173 9.184 R950174 9999 R950175 9999 R950176 9999 R950177 9999 R950178 22.59 R950179 29.867 R950180 2.869 R950181 2.689 R950182 9999 R950183 9999 R950184 9999 R950185 9999 R950186 5.944 R950187 22.312 R950188 17.862 R950189 21.963 R950190 7.17 R950191 2.586 R950192 1.732 R950193 2.826 R950194 5.131 R950195 1.804 R950196 2.081 R950197 2.582 R950198 1.99 R950199 3.214 R950200 2.264 R950201 4.502 R950202 9999 R950203 9999 R950204 9999 R950205 24.548 R950206 9999 R950207 1.085 R950208 1.766 R950209 3.796 R950210 9999 R950211 9999 R950212 9.497 R950213 9999 R950214 9999 R950215 5.006 R950216 3.856 R950217 2.795 R950218 3.425 R950219 2.11 R950220 2.678 R950221 20.345 R950222 2.008 R950223 2.775 R950224 2.423 R950225 2.325 R950226 2.917 R950227 7.112 R950229 3.773 R950230 8.235 R950231 8.688 R950232 9.161 R950233 5.305 R950234 9999 R950235 6.262 R950236 9.693 R950237 12.901 R950238 9999 R950239 9999 R950240 8.925 R950241 5.185 R950244 9999 R950245 9999 R950246 9999 R950247 9999 R950251 9999 R950253 10.547 R950254 2.35 R950255 9999 R950261 17.375 R950262 3.148 R950263 1.911 R950264 1.988 R950265 0.982 R950266 3.66 R950267 1.985 R950290 9999 R950291 9999 R950292 9999 R950293 9999 R950294 9.793 R950295 4.713 R950296 1.947 R950344 9999 R950345 6.09 R950346 1.948 R950347 2.704 R950348 0.224 R950349 0.363 R950356 5.731 R950368 0.125 R950371 1.105 R950372 2.192 R950373 3.614 R950374 1.65 R950376 18.08 R950377 5.962 R950378 9999 R950379 0.878 R950380 8.688 R950381 0.805 R950382 1.547 R950383 1.026 R950385 2.58 R950386 11.354

Although the foregoing invention has been described in some detail to facilitate understanding, it will be apparent that certain changes and modifications may be practiced within the scope of the appended claims. Accordingly, the described embodiments are to be considered as illustrative and not restrictive, and the invention is not to be limited to the details given herein, but may be modified within the scope and equivalents of the appended claims.

All literature and patent references cited throughout the application are incorporated by reference into the application for all purposes.

LENGTHY TABLES The patent application contains a lengthy table section. A copy of the table is available in electronic form from the USPTO web site (http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20120230984A1). An electronic copy of the table will also be available from the USPTO upon request and payment of the fee set forth in 37 CFR 1.19(b)(3). 

1-14. (canceled)
 15. A method of treating an autoimmune disease and/or one or more symptoms associated therewith in a mammal, the method comprising administering to the mammal an effective amount of a compound of formula (I) and at least one additional compound selected from a steroid, a membrane stabilizer, a 5LO inhibitor, a leukotriene synthesis inhibitor, a leukotriene receptor inhibitor, an inhibitor of IgE isotype switching, an inhibitor of IgE synthesis, an inhibitor of IgG isotype switching, an inhibitor of IgG synthesis, a β-agonist, a tryptase inhibitor, aspirin, a COX inhibitor, methotrexate, an anti-TNF drug, rituxan, a PD4 inhibitor, a p38 inhibitor, a PDE4 inhibitor, an antihistamine, and a combination thereof, wherein the compound of formula (I) is:

and salts, hydrates, solvates and N-oxides thereof, wherein: L¹ and L² are each a direct bond; R² is selected from the group consisting of phenyl mono-substituted at the 3- or 5-position with an R⁸ group, phenyl di or tri-substituted with one or more of the same or different R⁸ group, and 5-15 membered heteroaryl optionally substituted with one or more of the same or different R⁸ groups; R⁴ is selected from the group consisting of phenyl substituted with one or more of the same or different R⁸ groups, and 5-15 membered heteroaryl optionally substituted with one or more of the same or different R⁸ groups; R⁵ is selected from the group consisting of —CN, —NC, —NO₂, fluoro, (C1-C3) haloalkyl, (C1-C3) perhaloalkyl, (C1-C3) haloalkoxy, (C1-C3) perhaloalkoxy, —C(O)R^(a), —C(O)OR^(a), —C(O)CF₃ and —C(O)OCF₃; R⁶ is hydrogen; R⁸ is selected from the group consisting of R^(e), R^(b), R^(e) substituted with one or more of the same or different R^(a) or R^(b), —OR^(a) substituted with one or more of the same or different R^(a) or R^(b), —B(OR^(a))₂, —B(NR^(c)R^(c))₂, —(CH₂)_(m)—R^(b), —(CHR^(a))_(m)—R^(b), —O—(CH₂)_(m)—R_(b), —S—(CH₂)_(m)—R^(b), —O—CHR^(a)R^(b), —O—CR^(a)(R^(b))₂, —O—(CHR^(a))_(m)—R^(b), —O—(CH₂)_(m)—CH[(CH₂)_(m)R^(b)]R^(b), —S—(CHR^(a))_(m)—R^(b), —C(O)NH—(CH₂)_(m)—R^(b), —C(O)NH—(CHR^(a))_(m)—R^(b), —O—(CH₂)_(m)—C(O)NH—(CH₂)_(m)—R^(b), —S—(CH₂)_(m)—C(O)NH—(CH₂)_(m)—R^(b), —O—(CHR^(a))_(m)—C(O)NH—(CHR^(a))_(m)—R^(b), —S—(CHR^(a))_(m)—C(O)NH—(CHR^(a))_(m)—R^(b), —NH—(CH₂)_(m)—R^(b), —NH—(CHR^(a))_(m)—R^(b), —NH[(CH₂)_(m)R^(b)], —N[(CH₂)_(m)R^(b)]₂, —NH—C(O)—NH—(CH₂)_(m)—R^(b), —NH—C(O)—(CH₂)_(m)—CHR^(b)R^(b) and —NH—(CH₂)_(m)—C(O)—NH—(CH₂)_(m)—R^(b); each R^(a) is independently selected from the group consisting of hydrogen, (C1-C6) alkyl, (C3-C8) cycloalkyl, (C4-C11) cycloalkylalkyl, (C5-C10) aryl, (C6-C16) arylalkyl, 2-6 membered heteroalkyl, 3-8 membered cycloheteroalkyl, 4-11 membered cycloheteroalkylalkyl, 5-10 membered heteroaryl and 6-16 membered heteroarylalkyl; each R^(b) is a suitable group independently selected from the group consisting of ═O, —OR^(d), (C1-C3) haloalkyloxy, ═S, —SR^(d), ═NR^(d), ═NOR^(d), —NR^(c)R^(c), halogen, —CF₃, —CN, —NC, —OCN, —SCN, —NO, —NO₂, ═N₂, —N₃, —S(O)R^(d), —S(O)₂R^(d), —S(O)₂OR^(d), —S(O)NR^(c)R^(c), —S(O)₂NR^(c)R^(c), —OS(O)R^(d), —OS(O)₂R^(d), —OS(O)₂OR^(d), —OS(O)₂NR^(c)R^(c), —C(O)R^(d), —C(O)OR^(d), —C(O)NR^(c)R^(c), —C(NH)NR^(c)R^(c), —C(NR^(a))NR^(c)R^(c), —C(NOH)R^(a), —C(NOH)NR^(c)R^(c), —OC(O)R^(d), —OC(O)OR^(d), —OC(O)NR^(c)R^(c), —OC(NH)NR^(c)R^(c), —OC(NR^(a))NR^(c)R^(c), —[NHC(O)]_(n)R^(d), —[NR^(a)C(O)]_(n)R^(d), —[NHC(O)]_(n)OR^(d), —[NR^(a)C(O)]_(n)OR^(d), —[NHC(O)]_(n)NR^(c)R^(c), —[NR^(a)C(O)_(n)NR^(c)R^(c), —[NHC(NH)_(n)NR^(c)R^(c) and —[NR^(a)C(NR^(a))_(n)NR^(c)R^(c); each R^(c) is independently a protecting group or R^(a), or, alternatively, two R^(c) are taken together with the nitrogen atom to which it is bonded to form a 5 to 8-membered cycloheteroalkyl or heteroaryl which may optionally include one or more of the same or different additional heteroatoms and which may optionally be substituted with one or more of the same or different R^(a) or suitable R^(b) groups; each R^(d) is independently an R^(a); each R^(e) is independently selected from the group consisting of (C1-C6) alkyl, (C3-C8) cycloalkyl, (C4-C11) cycloalkylalkyl, (C5-C10) aryl, (C6-C16) arylalkyl, 2-6 membered heteroalkyl, 3-8 membered cycloheteroalkyl, 4-11 membered cycloheteroalkylalkyl, 5-10 membered heteroaryl, and 6-16 membered heteroarylalkyl; each m is independently an integer from 1 to 3; and each n is independently an integer from 0 to 3, wherein one or both of R² and R⁴, independently of one another, is an optionally substituted heteroaryl selected from the group consisting of:

wherein: p is an integer from one to three; each - - - independently represents a single bond or a double bond; R³⁵ is hydrogen or R⁸; X is selected from the group consisting of CH, N and N—O; each Y is independently selected from the group consisting of O, S and NH; each Y¹ is independently selected from the group consisting of O, S, SO, SO₂, SONR³⁶, NH and NR³⁷; each Y² is independently selected from the group consisting of CH, CH₂, O, S, N, NH and NR³⁷; R³⁶ is hydrogen or alkyl; R³⁷ is selected from the group consisting of hydrogen, aryl, arylalkyl, heteroaryl, R^(a), R^(b)—CR^(a)R^(b)—O—C(O)R⁸, —CR^(a)R^(b)—O—PO(OR⁸)₂, —CH₂—O—PO(OR⁸)₂, —CH₂—PO(OR⁸)₂, —C(O)—CR^(a)R^(b)—N(CH₂)₂, —CR^(a)R^(b)—O—C(O)—CR^(a)R^(b)—N(CH₃)₂, —C(O)R⁸, —C(O)CF₃ and —C(O)—NR⁸—C(O)R⁸; R³⁸ is selected from the group consisting of alkyl and aryl; A is selected from the group consisting of O, NH and NR³⁸; R⁹, R¹⁰, R¹¹ and R¹² are each, independently of one another, selected from the group consisting of alkyl, alkoxy, halogen, haloalkoxy, aminoalkyl and hydroxyalkyl, or, alternatively, R⁹ and R¹⁰ and/or R¹¹ and R¹² are taken together form an oxo group; each Z is selected from the group consisting of hydroxyl, alkoxy, aryloxy, ester, and carbamate; Q is selected from the group consisting of —OH, —OR⁸, —NR^(c)R^(c), —NHR³⁹—C(O)R⁸, —NHR³⁹—C(O)OR⁸, —NR³⁹—CHR⁴⁰—R^(b), and —NR³⁹—(CH₂)_(m)—R^(b); R³⁹ and R⁴⁰ are each, independently of one another, selected from the group consisting of hydrogen, alkyl, aryl, and arylalkyl.
 16. The method of claim 15 wherein the mammal is a human.
 17. (canceled)
 18. The method of claim 16, wherein the autoimmune disease is selected from the group consisting of Hashimoto's thyroiditis, autoimmune hemolytic anemia, autoimmune atrophic gastritis of pernicious anemia, autoimmune encephalomyelitis, autoimmune orchitis, Goodpasture's disease, autoimmune thrombocytopenia, sympathetic ophthalmia, myasthenia gravis, Graves' disease, primary biliary cirrhosis, chronic aggressive hepatitis, ulcerative colitis and membranous glomerulopathy.
 19. The method of claim 16, wherein the autoimmune disease is selected from the group consisting of systemic lupus erythematosis, rheumatoid arthritis, Sjogren's syndrome, Reiter's syndrome, polymyositis-dermatomyositis, systemic sclerosis, polyarteritis nodosa, multiple sclerosis and bullous pemphigoid.
 20. The method of claim 16 wherein the autoimmune disease is systemic lupus erythematosis. 21-30. (canceled)
 31. The method of claim 15, wherein the autoimmune disease is rheumatoid arthritis.
 32. The method of claim 15, wherein the one or more additional compounds comprises methotrexate, an anti-TNF drug or a combination thereof.
 33. The method of claim 32, wherein the one or more additional compounds includes methotrexate.
 34. The method of claim 32, wherein the one or more additional compounds includes an anti-TNF drug.
 35. The method of claim 15 in which R² is selected from the group consisting of phenyl, benzodioxanyl, benzodioxolyl, benzoxazinyl, benzoxazolyl, benzopyranyl, benzotriazolyl, 1,4-benzoxazinyl-2-one, 2H-1,4-benzoxazinyl-3(4H)-one, 2H-1,3-benzoxazinyl-2,4(3H)-dione, benzoxazolyl-2-one, dihydrocoumarinyl, 1,2-benzopyronyl, benzofuranyl, benzo[b]furanyl, indolyl, and pyrrolyl, each of which may be optionally substituted with one or more of the same or different R⁸ groups.
 36. The method of claim 15 in which R² and R⁴ are the same.
 37. The method of claim 36 in which each R³⁵ is independently selected from the group consisting of hydrogen, R^(d), —NR^(c)R^(c), —(CH₂)_(m)—NR^(c)R^(c), —C(O)NR^(c)R^(c), —(CH₂)_(m)—C(O)NR^(c)R^(c), —C(O)OR^(d), —(CH₂)_(m)—C(O)OR^(d) and —(CH₂)_(m)—OR^(d).
 38. The method of claim 37 in which each m is one.
 39. The method of claim 15 in which R² is an optionally substituted 5-15 membered heteroaryl which is attached to the remainder of the molecule via a ring carbon atom.
 40. The method of claim 15 in which R⁴ is an optionally substituted 5-15 membered heteroaryl which is attached to the remainder of the molecule via a ring carbon atom.
 41. The method of claim 15 in one of R² or R⁴ is a phenyl optionally substituted with one, two or three R⁸ groups.
 42. The method of claim 41 in which the optionally substituted phenyl is mono substituted.
 43. The method of claim 42 in which the R⁸ substituent is at the ortho, meta or para position.
 44. The method of claim 43 wherein R⁸ is selected from the group consisting of (C1-C10) alkyl, (C₁-C₁₀) branched alkyl, —OR^(d), —O—(CH₂)_(m)—NR^(c)R^(c), —O—C(O)NR^(c)R^(c), —O—(CH₂)_(m)—C(O)NR^(c)R^(c), —O—C(O)OR^(a), —O—(CH₂)_(m)—C(O)OR^(a), —O—C(NH)NR^(c)R^(c), —O—(CH₂)_(m)C(NH)NR^(c)R^(c), —NH—(CH₂)_(m)—NR^(c)R^(c), —NH—C(O)NR^(c)R^(c), and —NH—(CH₂)_(m)—C(O)NR^(c)R^(c).
 45. The method of claim 41 in which the optionally substituted phenyl is a disubstituted phenyl.
 46. The method of claim 45 in which the R⁸ substituents are positioned 2,3-; 2,4-; 2,5-; 2,6-; 3,4-; or 3,5-.
 47. The method of claim 45 in which each R⁸ is independently selected from the group consisting of (C1-C10) alkyl, (C1-C10) branched alkyl, —OR^(a) optionally substituted with one or more of the same or different R^(a) or R^(b) groups, —O—(CH₂)_(m)—NR^(c)R^(c), —O—C(O)NR^(c)R^(c), —O—(CH₂)_(m)—C(O)NR^(c)R^(c), —O—C(O)OR^(a), —O—(CH₂)_(m)—C(O)OR^(a), —O—C(NH)NR^(c)R^(c), —O—(CH₂)_(m)—C(NH)NR^(c)R^(c), —NH—(CH₂)_(m)—NR^(c)R^(c), —NH—C(O)NR^(c)R^(c), and —NH—(CH₂)_(m)—C(O)NR^(c)R^(c).
 48. The method of claim 41 in which the optionally substituted phenyl is trisubstituted.
 49. The method of claim 48 in which the R⁸ substituents are positioned 2,3,4-; 2,3,5-; 2,3,6-; 2,4,5-; 2,4,6-; 2,5,6-; or 3,4,5-.
 50. The method of claim 49 which each R⁸ is independently selected from the group consisting of (C1-C10) alkyl, (C1-C10) branched alkyl, —OR^(a) optionally substituted with one or more of the same or different R^(a) or R^(b) groups, —O—(CH₂)_(m)—NR^(c)R^(c), —O—C(O)NR^(c)R^(c), —O—(CH₂)_(m)—C(O)NR^(c)R^(c), —O—C(O)OR^(a), —O—C(NH)NR^(c)R^(c), —O—(CH₂)_(m)—C(O)OR^(a), —O—(CH₂)_(m)—C(NH)NR^(c)R^(c), —NH—(CH₂)_(m)—NR^(c)R^(c), —NH—C(O)NR^(c)R^(c), and —NH—(CH₂)_(m)—C(O)NR^(c)R^(c).
 51. The method of claim 48 in which the trisubstituted phenyl has the formula:

wherein: R³¹ is methyl or (C1-C6) alkyl; R³² is hydrogen, methyl or (C1-C6) alkyl; and R³³ is a halo group.
 52. The method of claim 15 in which the compound of formula (I) is a compound according to structural formula (Ic):

or a salt, hydrate, solvate and/or N-oxide thereof, wherein: R⁴ is an optionally substituted heteroaryl; and R¹⁸ is —O(CH₂)_(m)—R^(b).
 53. The method of claim 51 in which R⁸ is —O—CH₂—C(O)—NHCH₃.
 54. The method of claim 32 wherein the methotrexate is administered in an amount of from about 7.5 to about 25 mg/week.
 55. The method of claim 15 in which the compound of formula (I) is selected from the group consisting of: N4-[(2,2-Dimethyl-4H-benzo[1,4]oxazin-3-one)-6-yl]-5-fluoro-N2-[3-(methylaminocarbonylmethyleneoxy)phenyl]-2,4-pyrimidinediamine; N4-[(2,2-Dimethyl-4H-5-pyrido[1,4]oxazin-3-one)-6-yl]-5-fluoro-N243-(methylaminocarbonylmethyleneoxy)phenyl]-2,4-pyrimidinediamine; N4-[(2,2-Difluoro-4H-benzo[1,4]oxazin-3-one)-6-yl]-5-fluoro-N243-(methylaminocarbonylmethyleneoxy)phenyl]-2,4-pyrimidinediamine; N4-(2-Aminopyrid-6-yl)-5-fluoro-N243-(N-methylamino)carbonylmethyleneoxyphenyl]-2,4-pyrimidinediamine; N4-[2-(N-Acetylamino)pyrid-6-yl]-5-fluoro-N243-(N-methylamino)carbonylmethyleneoxyphenyl]-2,4-pyrimidinediamine; N2-(3,5-Dichloro-4-hydroxyphenyl)-N4-(2,2-difluoro-3-oxo-4H-benz[1,4]oxazin-6-yl)-5-fluoro-2,4-pyrimidinediamine; N2-(3,5-Dichloro-4-hydroxyphenyl)-N4-(2,2-dimethyl-3-oxo-4H-benz[1,4]oxazin-6-yl)-5-fluoro-2,4-pyrimidinediamine; N2-(3,5-Dichloro-4-methoxyphenyl)-N4-(2,2-dimethyl-3-oxo-4H-benz[1,4]oxazin-6-yl)-5-fluoro-2,4-pyrimidinediamine; N2-(3,5-Dichloro-4-methoxyphenyl)-N4-(2,2-difluoro-3-oxo-4H-benz[1,4]oxazin-6-yl)-5-fluoro-2,4-pyrimidinediamine; N2-(3,5-Dimethoxyphenyl)-N4-(2,2-dimethyl-3-oxo-4H-benz[1,4]oxazin-3-6-yl)-5-fluoro-2,4-pyrimidinediamine; N4-[2-Aminopyrid-6-yl)-N2-(3,5-dimethoxyphenyl)-5-fluoro-2,4-pyrimidinediamine; N2-(3,5-Dimethoxyphenyl)-5-fluoro-N4-(indol-6-yl)-2,4-pyrimidinediamine; N4-[2-(N-Acetylamino)pyrid-6-yl]-N2-(3,5-dimethoxyphenyl)-5-fluoro-2,4-pyrimidinediamine; N2-(3,5-Dichlorophenyl)-N4-(2,2-dimethyl-3-oxo-4H-benz[1,4]oxazin-6-yl)-5-fluoro-2,4-pyrimidinediamine; N4-(2,2-Dimethyl-3-oxo-4H-benz[1,4]oxazin-6-yl)-5-fluoro-N2-(3-methoxy-5-trifluoromethylphenyl)-2,4-pyrimidinediamine; N2-(2,6-Dimethoxypyrid-3-yl)-N4-(2,2-dimethyl-3-oxo-4H-benz[1,4]oxazin-6-yl)-5-fluoro-2,4-pyrimidinediamine; N4-[2-(N-Acetylamino)pyrid-6-yl]-N2-(3,5-dimethylphenyl)-5-fluoro-2,4-pyrimidinediamine; N2-(3,5-Dimethylphenyl)-5-fluoro-N4-[2-(N-methylamino)carbonylaminopyrid-6-yl]-2,4-pyrimidinediamine; N4-(2-Aminopyrid-6-yl)-N2-(3-chloro-4-trifluoromethoxyphenyl)-5-fluoro-2,4-pyrimidinediamine; N2-(3-Chloro-4-trifluoromethoxyphenyl)-N4-(3,4-ethylenedioxyphenyl)-5-fluoro-2,4-pyrimidinediamine; N4-(2-Aminopyrid-6-yl)-N2-(3-chloro-4-methoxyphenyl)-5-fluoro-2,4-pyrimidinediamine; N2-(3-Chloro-4-methoxyphenyl)-N4-(3,4-ethylenedioxyphenyl)-5-fluoro-2,4-pyrimidinediamine; N2-(3-Chloro-4-methoxyphenyl)-5-fluoro-N4-(indol-6-yl)-2,4-pyrimidinediamine; N4-(2-Aminopyrid-6-yl)-5-fluoro-N2-(3-hydroxyphenyl)-2,4-pyrimidinediamine; N2-(3-Chloro-4-methoxyphenyl)-N4-(2,2-dimethyl-3-oxo-4H-benz[1,4]oxazin-6-yl)-5-fluoro-2,4-pyrimidinediamine; N4-(3,4-Dichlorophenyl)-N2-(2,2-dimethyl-3-oxo-4H-benz[1,4]oxazin-6-yl)-5-fluoro-2,4-pyrimidinediamine; N2-(4-Chloro-3,5-dimethylphenyl)-N4-(3,4-ethylenedioxyphenyl)-5-fluoro-2,4-pyrimidinediamine; N2-(4-Chloro-3,5-dimethylphenyl)-N4-(2,2-dimethyl-3-oxo-4H-benz[1,4]oxazin-6-yl)-5-fluoro-2,4-pyrimidinediamine; (±)-N-4-(2,2-Difluoro-3-oxo-4H-benz[1,4]oxazin-6-yl)-N2-[2-(N-methylaminocarbonyl)-2,3-dihydrobenzofuran-5-yl]-5-fluoro-2,4-pyrimidinediamine; (±)-N4-(3-Chloro-4-methoxyphenyl)-N2-[2-(N,N-dimethylaminocarbonyl)-2,3-dihydrobenzofuran-5-yl]-5-fluoro-2,4-pyrimidinediamine; (±)-N4-(3-Chloro-4-trifluoromethoxyphenyl)-N242-(N,N-dimethylaminocarbonyl)-2,3-dihydrobenzofuran-5-yl]-5-fluoro-2,4-pyrimidinediamine; (±)-N4-(3-Chloro-4-methoxyphenyl)-N2-[2-(N-methylaminomethylene)-2,3-dihydrobenzofuran-5-yl]-5-fluoro-2,4-pyrimidinediamine; 5-Fluoro-N2-[2(R)-{(1R,2S,5R)-menthyloxycarbonyl}-2,3-dihydrobenzofuran-5-yl]-N4-(4-isopropoxyphenyl)-2,4-pyrimidinediamine; N4-(2,2-Difluoro-3-oxo-4H-benz[1,4]oxazin-6-yl)-N2-[2(R)-{(1R,2S,5R)-menthyloxycarbonyl}-2,3-dihydrobenzofuran-5-yl]-5-fluoro-2,4-pyrimidinediamine; N2-(3,5-Dimethoxyphenyl)-N4-(2,2-dimethyl-3-oxo-4H-benz[1,4]oxazin-6-yl)-5-fluoro-2,4-pyrimidinediamine p-Toluenesulfonic Acid Salt; N2-(3,5-Dimethoxyphenyl)-N4-(2,2-dimethyl-3-oxo-4H-benz[1,4]oxazin-6-yl)-5-fluoro-2,4-pyrimidinediamine Methanesulfonic Acid Salt; N2-(3,5-Dimethoxyphenyl)-N4-(2,2-dimethyl-3-oxo-4H-benz[1,4]oxazin-6-yl)-5-fluoro-2,4-pyrimidinediamine Benzenesulfonic Acid Salt; N2-(3,5-Dimethoxyphenyl)-N4-(2,2-dimethyl-3-oxo-4H-benz[1,4]oxazin-6-yl)-5-fluoro-2,4-pyrimidinediamine Hydrogen Chloride Salt; N2-(3,5-Dimethoxyphenyl)-N4-(2,2-dimethyl-3-oxo-4H-benz[1,4]oxazin-6-yl)-5-fluoro-2,4-pyrimidinediamine DL-Camphoursulfonic Acid Salt; N2-(3,5-Dimethylphenyl)-N4-(2,2-dimethyl-3-oxo-4H-benz[1,4]oxazin-6-yl)-5-fluoro-2,4-pyrimidinediamine p-Toluenesulfonic Acid Salt; N2-(3,5-Dimethylphenyl)-N4-(2,2-dimethyl-3-oxo-4H-benz[1,4]oxazin-6-yl)-5-fluoro-2,4-pyrimidinediamine Benzenesulfonic Acid Salt; N4-(3-Chloro-4-methoxyphenyl)-5-fluoro-N2-(indol-6-yl)-2,4-pyrimidinediamine p-Toluenesulfonic Acid Salt; N4-(3,4-Ethylendioxyphenyl)-5-fluoro-N2-[3-(N-methylamino)carbonylmethyleneoxyphenyl]-2,4-pyrimidinediamine p-Toluenesulfonic Acid Salt; (±) N2-(2-Carboxyl-2,3-dihydrobenzofuran-5-yl)-N4-(3-chloro-4-trifluoromethoxyphenyl)-5-fluoro-2,4-pyrimidinediamine; (±) N4-(3-Chloro-4-trifluoromethoxyphenyl)-5-fluoro-N2-[2-(N-2,3-dihydroxypropylamino)carbonyl-2,3-dihydrobenzofuran-5-yl]-2,4-pyrimidinediamine; (±) N4-(3-Chloro-4-trifluoromethoxyphenyl)-5-fluoro-N2-[2-(N-2-hydroxyethylamino)carbonyl-2,3-dihydrobenzofuran-5-yl]-2,4-pyrimidinediamine; (±) N4-(3-Chloro-4-trifluoromethoxyphenyl)-5-fluoro-N2-[2-(N2-hydroxyethyl-N-methylamino]carbonyl-2,3-dihydrobenzofuran-5-yl]-2,4-pyrimidinediamine; N4-(3-Chloro-4-trifluoromethoxyphenyl)-5-fluoro-N2-[2-(N-isopropylamino)carbonyl-2,3-dihydrobenzofuran-5-yl]-2,4-pyrimidinediamine; 5-Fluoro-N4-(2-isopropoxypyridin-5-yl)-N243-(N-methylamino)carbonylmethyleneoxyphenyl]-2,4-pyrimidinediamine; N4-(2,2-Difluoro-3-oxo-4H-benz[1,4]oxazin-6-yl)-N2-(3,5-dimethoxyphenyl)-5-fluoro-2,4-pyrimidinediamine; N2-(3-Chloro-4-hydroxy-5-methylphenyl)-N4-(2,2-difluoro-3-oxo-4H-benz[1,4]oxazin-6-yl)-5-fluoro-2,4-pyrimidinediamine; N4-(3,4-Dichlorophenyl)-5-fluoro-N2-(indol-6-yl)-2,4-pyrimidinediamine; N4-(3-Chloro-4-methoxyphenyl)-5-fluoro-N2-(indol-6-yl)-2,4-pyrimidinediamine; N4-(2,2-Difluoro-3-oxo-4H-benz[1,4]oxazin-6-yl)-5-fluoro-N2-(indol-6-yl)-2,4-pyrimidinediamine; N4-(3-Chloro-4-methoxyphenyl)-N242-(N,N-dimethylaminomethyl)benzofuran-5-yl]-5-fluoro-2,4-pyrimidinediamine; N4-(2,2-Difluoro-3-oxo-4H-benz[1,4]oxazin-6-yl)-N2-[2-(N,N-dimethylaminomethyl)benzofuran-5-yl]-5-fluoro-2,4-pyrimidinediamine; N4-(3,4-Dichlorophenyl)-N2-[2-(N,N-dimethylaminomethyl)benzofuran-5-yl]-5-fluoro-2,4-pyrimidinediamine; N2-(3-Chloro-4-methoxyphenyl)-N4-(2,2-difluoro-3-oxo-4H-benz[1,4]oxazin-6-yl)-5-fluoro-2,4-pyrimidinediamine; N4-(2,2-Dimethyl-3-oxo-4H-benz[1,4]oxazin-6-yl)-5-fluoro-N2-(indol-6-yl)-2,4-pyrimidinediamine; N4-(2,2-Difluoro-3-oxo-4H-benz[1,4]oxazin-6-yl)-N242-(N,N-dimethylaminocarbonyl)-2,3-dihydrobenzofuran-5-yl]-5-fluoro-2,4-pyrimidinediamine; N4-(2,2-Difluoro-3-oxo-4H-benz[1,4]oxazin-6-yl)-5-fluoro-N2-(3-methoxy-5-trifluoromethylphenyl)-2,4-pyrimidinediamine; N2-(3,5-Dichlorophenyl)-N4-(2,2-difluoro-3-oxo-4H-benz[1,4]oxazin-6-yl)-5-fluoro-2,4-pyrimidinediamine; N4-(2,2-Dimethyl-3-oxo-4H-benz[1,4]oxazin-6-yl)-5-fluoro-N2-(3,4,5-trimethylphenyl)-2,4-pyrimidinediamine; N2-(3-Chloro-4-hydroxy-5-methylphenyl)-N4-(2,2-dimethyl-3-oxo-4H-benz[1,4]oxazin-6-yl)-5-fluoro-2,4-pyrimidinediamine; N2-(3-Chloro-4-methoxy-5-methylphenyl)-N4-(2,2-dimethyl-3-oxo-4H-benz[1,4]oxazin-6-yl)-5-fluoro-2,4-pyrimidinediamine; N4-(2,2-Dimethyl-3-oxo-4H-benz[1,4]oxazin-6-yl)-5-fluoro-N2-(indazolin-6-yl)-2,4-pyrimidinediamine; N4-(2,2-Difluoro-3-oxo-4H-benz[1,4]oxazin-6-yl)-5-fluoro-N2-(indazolin-6-yl)-2,4-pyrimidinediamine; 5-Fluoro-N4-[2-methyl-2-(N-methylaminocarbonyl)-3-oxo-4H-benz[1,4]oxazin-6-yl]-N2-[3-(N-methylamino)carbonylmethyleneoxyphenyl]-2,4-pyrimidinediamine; N4-(2,2-Dimethyl-3-oxo-4H-benz[1,4]oxazin-6-yl)-5-fluoro-N2-(N1-methylindazolin-6-yl)-2,4-pyrimidinediamine; N4-(2,2-Difluoro-3-oxo-4H-benz[1,4]oxazin-6-yl)-N2-(2,2-dimethyl-3-oxo-4H-benz[1,4]oxazin-6-yl)-5-fluoro-2,4-pyrimidinediamine; N4-(2,2-Difluoro-3-oxo-4H-benz[1,4]oxazin-6-yl)-5-fluoro-N2-(1-methylindazolin-6-yl)-2,4-pyrimidinediamine; N4-(2,2-Dimethyl-3-oxo-4H-5-pyrid[1,4]oxazin-6-yl)-N2-(3-chloro-4-methoxyphenyl)-5-fluoro-2,4-pyrimidinediamine; N4-(2,2-Dimethyl-3-oxo-4H-5-pyrid[1,4]oxazin-6-yl)-N2-(3,5-dimethoxyphenyl)-5-fluoro-2,4-pyrimidinediamine; N2-(3,4-Dichlorophenyl)-N4-(2,2-dimethyl-3-oxo-4H-5-pyrid[1,4]oxazin-6-yl)-5-fluoro-2,4-pyrimidinediamine; N4-[(2,2-Dimethyl-3-oxo-4H-5-pyrid[1,4]oxazin-6-yl)-5-fluoro-N2-(indazolin-6-yl)-2,4-pyrimidinediamine; N2-(3-tert-Butylphenyl)-N4-(2,2-dimethyl-3-oxo-4H-5-pyrid[1,4]oxazin-6-yl)-5-fluoro-2,4-pyrimidinediamine; N4-[(2,2-Dimethyl-3-oxo-4H-5-pyrid[1,4]oxazin-6-yl)-5-fluoro-N2-(3-hydroxyphenyl)-2,4-pyrimidinediamine; N4-(2,2-dimethyl-3-oxo-4H-5-pyrid[1,4]oxazin-6-yl)-N2-(3-fluoro-4-methoxyphenyl)-5-fluoro-2,4-pyrimidinediamine; N2-(3-Chlorophenyl)-N4-(2,2-dimethyl-3-oxo-4H-5-pyrid[1,4]oxazin-6-yl)-5-fluoro-2,4-pyrimidinediamine; N2-(3,5-Dichlorophenyl)-N4-(2,2-dimethyl-3-oxo-4H-5-pyrid[1,4]oxazin-6-yl)-5-fluoro-2,4-pyrimidinediamine; N4-(2,2-Dimethyl-3-oxo-4H-5-pyrid[1,4]oxazin-6-yl)-5-fluoro-N2-(1-methylindazolin-6-yl)-2,4-pyrimidinediamine; N2-(3-Chloro-4-trifluoromethoxyphenyl)-N4-(2,2-dimethyl-3-oxo-4H-5-pyrid[1,4]oxazin-6-yl)-5-fluoro-2,4-pyrimidinediamine; N2-(3-Chloro-4-methoxy-5-methylphenyl)-N4-(2,2-dimethyl-3-oxo-4H-5-pyrid[1,4]oxazin-6-yl)-5-fluoro-2,4-pyrimidinediamine; N4-(2,2-Dimethyl-3-oxo-4H-5-pyrid[1,4]oxazin-6-yl)-5-fluoro-N2-(3-methoxycarbonylmethyleneoxyphenyl)-2,4-pyrimidinediamine; N2-(3-Chloro-4-hydroxy-5-methylphenyl)-N4-(3,4-ethylenedioxyphenyl)-5-fluoro-2,4-pyrimidinediamine; N2-(3-Chloro-4-hydroxy-5-methylphenyl)-N4-(2,2-dimethyl-3-oxo-4H-5-pyrid[1,4]oxazin-6-yl)-5-fluoro-2,4-pyrimidinediamine; N2-(3,5-Dimethyl-4-methoxyphenyl)-N4-(2,2-dimethyl-3-oxo-4H-5-pyrid[1,4]oxazin-6-yl)-5-fluoro-2,4-pyrimidinediamine; N2-(3,5-Dimethyl-4-methoxyphenyl)-N4-(3,4-ethylenedioxyphenyl)-5-fluoro-2,4-pyrimidinediamine; N2-(3-Chloro-4-methoxy-5-methylphenyl)-N4-(3,4-ethylenedioxyphenyl)-5-fluoro-2,4-pyrimidinediamine; (±) N4-[4-(N-tert-Butoxycarbonyl)amino-1-benzopyran-6-yl)-5-fluoro-N2-[3-(N-methylamino)carbonylmethyleneoxyphenyl]-2,4-pyrimidinediamine; (±) 5-Fluoro-N4-[4-(N-methyl)amino-1-benzopyran-6-yl]-N243-(N-methylamino)carbonylmethyleneoxyphenyl]-2,4-pyrimidinediamine; (±) N4-[4-(N-tert-Butoxycarbonyl-N-methyl)amino-1-benzopyran-6-yl]-5-fluoro-N243-(N-methylamino)carbonylmethyleneoxyphenyl]-2,4-pyrimidinediamine; (S)—N4-[4-(N-Benzyloxycarbonyl)amino-1-benzopyran-6-yl]-5-fluoro-N2-[3-(N-methylamino)carbonylmethyleneoxyphenyl]-2,4-pyrimidinediamine; (R)—N4-[4-(N-Benzyloxycarbonyl)amino-1-benzopyran-6-yl]-5-fluoro-N243-(N-methylamino)carbonylmethyleneoxyphenyl]-2,4-pyrimidinediamine; (S)—N4-(4-Amino-1-benzopyran-6-yl)-5-fluoro-N2-[3-(N-methylamino)carbonylmethyleneoxyphenyl]-2,4-pyrimidinediamine; (R)—N4-(4-Amino-1-benzopyran-6-yl)-5-fluoro-N2-[3-(N-methylamino)carbonylmethyleneoxyphenyl]-2,4-pyrimidinediamine; (±) N4-[4-(N-tert-Butoxycarbonyl)amino-1-benzopyran-6-yl]-5-fluoro-N2-(indazol-6-yl)-2,4-pyrimidinediamine; (±) N4-(4-Amino-1-benzopyran-6-yl)-5-fluoro-N2-(indazol-6-yl)-2,4-pyrimidinediamine; (±) N4-(4-Amino-1-benzopyran-6-yl)-N2-(3,5-dichloro-4-methoxyphenyl)-5-fluoro-2,4-pyrimidinediamine; (±) N4-[4-(N-tert-Butoxycarbonyl)amino-1-benzopyran-6-yl]-N2-(3,5-dimethoxyphenyl)-5-fluoro-2,4-pyrimidinediamine; (±) N4-(4-Amino-1-benzopyran-6-yl)-N2-(3,5-dimethoxyphenyl)-5-fluoro-2,4-pyrimidinediamine; (±) N4-[4-(N-tert-Butoxycarbonyl)amino-1-benzopyran-6-yl]-N2-(3-chloro-4-methoxyphenyl)-5-fluoro-2,4-pyrimidinediamine; (±) N4-(4-Amino-1-benzopyran-6-yl)-N2-(3-chloro-4-methoxyphenyl)-5-fluoro-2,4-pyrimidinediamine; (±) N4-[4-(N-tert-Butoxycarbonyl)amino-1-benzopyran-6-yl]-N2-(3,4-dichlorophenyl)-5-fluoro 2,4-pyrimidinediamine; (±) N4-(4-Amino-1-benzopyran-6-yl)-N2-(3,4-dichlorophenyl)-5-fluoro-2,4-pyrimidinediamine; (±) N2-[4-(N-tert-Butoxycarbonylamino-1-benzopyran-6-yl]-N4-(3,5-dimethoxyphenyl)-5-fluoro-2,4-pyrimidinediamine; (±) N2-[4-(N-tert-Butoxycarbonyl)amino-1-benzopyran-6-yl]-N4-(3,4-dichlorophenyl)-5-fluoro-2,4-pyrimidinediamine; N2-[4(R,S)—(N-tert-Butoxycarbonyl)amino-1-benzopyran-6-yl]-5-fluoro-N4-[2-(S)-methyl-3-oxo-4H-benz[1,4]oxazin-6-yl]-2,4-pyrimidinediamine; (±) N2-(4-Amino-1-benzopyran-6-yl]-N4-(3,5-dimethoxyphenyl)-5-fluoro-2,4-pyrimidinediamine; (±) N2-(4-Amino-1-benzopyran-6-yl)-N4-(3,4-dichlorophenyl)-5-fluoro 2,4-pyrimidinediamine; N2-[4(R,S)-Amino-1-benzopyran-6-yl)-5-fluoro-N4-(2(S)-methyl-3-oxo-4H-benz[1,4]oxazin-6-yl]-2,4-pyrimidinediamine; (±)-5-Fluoro-N2-[3-(N-methylamino)carbonylmethyleneoxyphenyl]-N4-[4-(N-p-toluenesulfonyl)amino-1-benzopyran-6-yl)-2,4-pyrimidinediamine; (±)-5-Fluoro N4-[4-(N-methanesulfonyl)amino-1-benzopyran-6-yl)-N2-[3-(N-methylamino)carbonylmethyleneoxyphenyl]-2,4-pyrimidinediamine; (±)—N4-[4-N—(N,N-Dimethylaminomethylencarbonyl)amino-1-benzopyran-6-yl]-5-fluoro-N2-[3-(N-methylamino)carbonylmethyleneoxyphenyl]-2,4-pyrimidinediamine; (±)—N4-[4-N—(N,N-Dimethylaminomethylencarbonyl)-N-methylamino-1-benzopyran-6-yl]-5-fluoro-N2-[3-(N-methylamino)carbonylmethyleneoxyphenyl]-2,4-pyrimidinediamine; N2-[3-(N-Cyclobutylamino)carbonylmethyleneoxyphenyl]-5-fluoro-N4-(2H-3-oxo-4H-5-pyrid[1,4]oxazin-6-yl)-2,4-pyrimidinediamine; N2-[3-(N-Cyclopropylamino)carbonylmethyleneoxyphenyl]-5-fluoro-N4-(2H-3-oxo-4H-5-pyrid[1,4]oxazin-6-yl)-2,4-pyrimidinediamine; N2-(3,4-Dichlorophenyl)-5-fluoro-N4-(2H-3-oxo-4H-5-pyrid[1,4]oxazin-6-yl)-2,4-pyrimidinediamine; N2-(3-Chloro-4-methoxyphenyl)-5-fluoro-N4-(2H-3-oxo-4H-5-pyrid[1,4]oxazin-6-yl)-2,4-pyrimidinediamine; N2-(3,5-Dichloro-4-methoxyphenyl)-5-fluoro-N4-(2H-3-oxo-4H-5-pyrid[1,4]oxazin-6-yl)-2,4-pyrimidinediamine; N2-(3,5-Dimethoxyphenyl)-5-fluoro-N4-(2H-3-oxo-4H-5-pyrid[1,4]oxazin-6-yl)-2,4-pyrimidinediamine; 5-Fluoro-N2-(3-fluoro-4-methoxyphenyl)-N4-(2H-3-oxo-4H-5-pyrid[1,4]oxazin-6-yl)-2,4-pyrimidinediamine; N2-(3,4-Ethylenedioxyphenyl)-5-fluoro-N4-(2H-3-oxo-4H-5-pyrid[1,4]oxazin-6-yl)-2,4-pyrimidinediamine; N4-(2,2-Difluoro-2H-3-oxo-4H-benz[1,4]oxazin-6-yl)-5-fluoro-N2-[3-(N-methylamino)carbonylmethyleneoxyphenyl)-2,4-pyrimidinediamine p-Toluenesulfonic Acid Salt; N4-(2,2-Difluoro-2H-3-oxo-4H-benz[1.4]oxazin-6-yl)-5-fluoro-N2-[3-(N-methylamino)carbonylmethyleneoxyphenyl)-2,4-pyrimidinediamine Benzenesulfonic Acid Salt; N2-(3,4-Dimethoxyphenyl)-5-fluoro-N4-(2H-3-oxo-4H-5-pyrid[1,4]oxazin-6-yl)-2,4-pyrimidinediamine; N2-[3-(N-Cyclobutylamino)carbonylmethyleneoxyphenyl]-N4-(2,2-dimethyl-2H-3-oxo-4H-5-pyrid[1,4]oxazin-6-yl)-5-fluoro-2,4-pyrimidinediamine; N2-(3,5-Dimethylphenyl)-5-fluoro-N4-(2H-3-oxo-4H-5-pyrid[1,4]oxazin-6-yl)-2,4-pyrimidinediamine; N2-(3,5-Dimethylphenyl)-N4-(2,2-dimethyl-2H-3-oxo-4H-5-pyrid[1,4]oxazin-6-yl)-5-fluoro-2,4-pyrimidinediamine; 5-Fluoro-N2-(3-isopropylphenyl)-N4-(2H-3-oxo-4H-5-pyrid[1,4]oxazin-6-yl)-2,4-pyrimidinediamine; N2-(3-Chloro-4-methylphenyl)-5-fluoro-N4-(2H-3-oxo-4H-5-pyrid[1,4]oxazin-6-yl)-2,4-pyrimidinediamine; 5-Fluoro-N2-(3-methoxy-5-trifluoromethylphenyl)-N4-(2H-3-oxo-4H-5-pyrid[1,4]oxazin-6-yl)-2,4-pyrimidinediamine; 5-Fluoro-N2-(indol-6-yl)-N4-(2H-3-oxo-4H-5-pyrid[1,4]oxazin-6-yl)-2,4-pyrimidinediamine; N4-(2,2-dimethyl-2H-3-oxo-4H-5-pyrid[1,4]oxazin-6-yl)-5-fluoro-N2-(indol-6-yl)-2,4-pyrimidinediamine; N2-(3,5-Dichlorophenyl)-5-fluoro-N4-(2H-3-oxo-4H-5-pyrid[1,4]oxazin-6-yl)-2,4-pyrimidinediamine; N2-(3-Bromophenyl)-5-fluoro-N4-(2H-3-oxo-4H-5-pyrid[1,4]oxazin-6-yl)-2,4-pyrimidinediamine; N2-(3-tert-Butylphenyl)-5-fluoro-N4-(2H-3-oxo-4H-5-pyrid[1,4]oxazin-6-yl)-2,4-pyrimidinediamine; N2-(3,4-Difluorophenyl)-5-fluoro-N4-(2H-3-oxo-4H-5-pyrid[1,4]oxazin-6-yl)-2,4-pyrimidinediamine; (S)-5-Fluoro-N2-[3-(N-methylamino)carbonylmethyleneoxyphenyl]-N4-(2-methyl-3-oxo-4H-benz[1,4]oxazin-6-yl)-2,4-pyrimidinediamine; N4-(4,4-Dimethyl-1,3-dioxo-2H,4H-isoquinolin-7-yl)-5-fluoro-N2-[3-(N-methylamino)carbonylmethylenedioxyphenyl]-2,4-pyrimidinediamine; (R)-5-Fluoro-N2-[3-(N-methylamino)carbonylmethyleneoxyphenyl]-N4-(2-methyl-3-oxo-4H-benz[1,4]oxazin-6-yl)-2,4-pyrimidinediamine; N2-(3-Chloro-4-hydroxy-5-methylyphenyl)-N4-(4,4-dimethyl-1,3-dioxo-2H,4H-isoquinolin-7-yl)-5-fluoro-2,4-pyrimidinediamine; (S)—N2-(3-Chloro-4-methoxyphenyl)-5-fluoro-N4-(2-methyl-3-oxo-4H-benz[1,4]oxazin-6-yl)-2,4-pyrimidinediamine; (R)—N2-(3-Chloro-4-methoxyphenyl)-5-fluoro-N4-(2-methyl-3-oxo-4H-benz[1,4]oxazin-6-yl)-2,4-pyrimidinediamine; N2-(3,5-Dimethoxyphenyl)-N4-(4,4-dimethyl-1,3-dioxo-2H,4H-isoquinolin-7-yl)-5-fluoro-2,4-pyrimidinediamine; (S)—N2-(3,5-Dichloro-4-methoxyphenyl)-5-fluoro-N4-(2-methyl-3-oxo-4H-benz[1,4]-oxazin-6-yl)-2,4-pyrimidinediamine; (R)—N2-(3,5-Dichloro-4-methoxyphenyl)-5-fluoro-N4-(2-methyl-3-oxo-4H-benz[1,4]-oxazin-6-yl)-2,4-pyrimidinediamine; (±)-N2-(3,5-Dimethoxyphenyl)-5-fluoro-N4-[2-(2-hydroxyethyl)-3-oxo-4H-benz[1,4]oxazin-6-yl]-2,4-pyrimidinediamine; (±)-N2-(3-Chloro-4-methoxyphenyl)-5-fluoro-N4-[2-(2-hydroxyethyl)-3-oxo-4H-benz[1,4]oxazin-6-yl]-2,4-pyrimidinediamine; (S,S)—N2,N4-Bis-(2-methyl-3-oxo-4H-benz[1,4]oxazin-6-yl)-5-fluoro-2,4-pyrimidinediamine; (S)—N2-(3,5-Dimethoxyphenyl)-5-fluoro-N4-(2-methyl-3-oxo-4H-benz[1,4]oxazin-6-yl)-2,4-pyrimidinediamine; (R)—N2-(3,5-Dimethoxyphenyl)-5-fluoro-N4-(2-methyl-3-oxo-4H-benz[1,4]oxazin-6-yl)-2,4-pyrimidinediamine; N2-(3,5-Dichloro-4-methoxyphenyl)-N4-(4,4-dimethyl-1,3-dioxo-2H,4H-isoquinolin-7-yl)-5-fluoro-2,4-pyrimidinediamine; N4-(4,4-Dimethyl-1,3-dioxo-2H,4H-isoquinolin-7-yl)-N2-(indazol-6-yl)-5-fluoro-2,4-pyrimidinediamine; N4-(3,3-Dimethyl-4H-benz[1,4]oxazin-6-yl)-N2-(3,5-dimethylphenyl)-5-fluoro-2,4-pyrimidinediamine; N2-(3-Chloro-4-methoxyphenyl)-N4-(3,3-dimethyl-4H-benzo[1,4]oxazin-6-yl)-5-fluoro-2,4-pyrimidinediamine; N4-(3,3-Dimethyl-1,4-benzoxazin-6-yl)-5-fluoro-N2-(indazol-6-yl)-2,4-pyrimidinediamine; N4-(3,3-Dimethyl-4H-benz[1,4]oxazin-6-yl)-5-fluoro-N2-(N1-methylindazol-6-yl)-2,4-pyrimidinediamine; (R)—N2-(3-Chloro-4-methoxyphenyl)-5-fluoro-N4-(2-methyl-3-oxo-4H-benz[1,4]oxazin-6-yl)-2,4-pyrimidinediamine Toluenesulfonic Acid Salt; N4-(2,6-Dimethoxypyrid-3-yl)-N2-[1-(2-ethoxycarbonylethyl)indazolin-5-yl]-5-fluoro-2,4-pyrimidinediamine; N4-(4-Chlorophenyl)-5-fluoro-N2-{1-[2-(N-methylamino)carbonylethyl]indazolin-5-yl}-2,4-pyrimidinediamine; N4-(4-Chlorophenyl)-5-fluoro-N2-[1-(3-hydroxypropyl)indazolin-5-yl]-2,4-pyrimidinediamine; N4-(3,4-Difluorophenyl)-N2-[1-(2-ethoxycarbonylethyl)indazolin-5-yl]-5-fluoro-2,4-pyrimidinediamine; N4-(3,4-Difluorophenyl)-5-fluoro-N2-{1-[2-(N-methylaminocarbonyl)ethyl]indazolin-5-yl}-2,4-pyrimidinediamine; N4-(3,4-Difluorophenyl)-5-fluoro-N2-[1-(3-hydroxypropyl)indazolin-5-yl]-2,4-pyrimidinediamine; N4-(3,4-Dichlorophenyl)-N2-[1-(2-ethoxycarbonylethyl)indazolin-5-yl]-5-fluoro-2,4-pyrimidinediamine; N4-(3,4-Dichlorophenyl)-5-fluoro-N2-{1-[2(N-methylaminocarbonyl)ethyl]indazolin-5-yl}-2,4-pyrimidinediamine; N4-(3,4-Dichlorophenyl)-5-fluoro-N2-[1-(3-hydroxypropyl)indazolin-5-yl]-2,4-pyrimidinediamine; N4-(3,4-Ethylenedioxyphenyl)-5-fluoro-N2-(1-methylindazolin-6-yl)-2,4-pyrimidinediamine; N4-(3,4-Dichlorophenyl)-5-fluoro-N2-(1-methylindazolin-6-yl)-2,4-pyrimidinediamine; N2-(3-Chloro-4-hydroxy-5-methylphenyl)-5-fluoro-N4-(1-methylindazolin-6-yl)-2,4-pyrimidinediamine; N4-(3,4-Dichlorophenyl)-5-fluoro-N242-(2-methoxy-4-methoxycarbonylbenzyl)indazolin-6-yl]-2,4-pyrimidinediamine; N4-(3-Chloro-4-methoxyphenyl)-5-fluoro-N242-(2-methoxy-4-methoxycarbonylbenzyl)indazolin-6-yl]-2,4-pyrimidinediamine; N4-(3,4-Ethylenedioxyphenyl)-5-fluoro-N2-{1-[2-methoxy-4-(N-methylaminocarbonyl)benzyl]indazolin-6-yl}-2,4-pyrimidinediamine; N4-(3,4-Difluorophenyl)-5-fluoro-N2-(indazolin-6-yl)-2,4-pyrimidinediamine; N4-(3,4-Difluorophenyl)-5-fluoro-N2-(1-methylindazolin-6-yl)-2,4-pyrimidinediamine; N4-(3-Chloro-4-methoxyphenyl)-5-fluoro-N2-(1-methylindazolin-6-yl)-2,4-pyrimidinediamine; N4-(3,4-Dichlorophenyl)-N2-[1-(2-ethoxycarbonylethyl)indazolin-6-yl]-5-fluoro-2,4-pyrimidinediamine; N4-(3,4-Dichlorophenyl)-5-fluoro-N2-{1-[2(N-methylamino)carbonylethyl]indazolin-6-yl}-2,4-pyrimidinediamine; N4-(3,4-Dichlorophenyl)-5-fluoro-N2-[1-(3-hydroxypropyl)indazolin-6-yl]-2,4-pyrimidinediamine; N4-(3-Chloro-4-methoxyphenyl)-N2-[1-(2-ethoxycarbonylethyl)indazolin-6-yl]-5-fluoro-2,4-pyrimidinediamine; N4-(3-Chloro-4-methoxyphenyl)-5-fluoro-N2-[1-(3-hydroxypropyl)indazolin-6-yl]-2,4-pyrimidinediamine; N4-(3-Chloro-4-methoxyphenyl)-5-fluoro-N2-(indazolin-6-yl)-2,4-pyrimidinediamine; N4-(3-Chloro-4-methoxyphenyl)-5-fluoro-N2-{1-[2-(N-methylaminocarbonyl)ethyl]indazolin-6-yl}-2,4-pyrimidinediamine; 5-Fluoro-N4-(4-fluoro-3-methoxyphenyl)-5-fluoro-N2-(1-methylindazolin-5-yl)-2,4-pyrimidinediamine; N4-(4-Chloro-3-fluorophenyl)-5-fluoro-N2-(1-methylindazolin-5-yl)-2,4-pyrimidinediamine; N4-(3,4-Dimethoxyphenyl)-5-fluoro-N2-(1-methylindazolin-5-yl)-2,4-pyrimidinediamine; N2-(3-Chloro-4-methoxy-5-methylphenyl)-5-fluoro-N4-(indazolin-6-yl)-2,4-pyrimidinediamine; N4-(4-Chloro-3-fluorophenyl)-5-fluoro-N2-(indazolin-6-yl)-2,4-pyrimidinediamine; N4-(4-Chloro-3-fluorophenyl)-5-fluoro-N2-(indazolin-5-yl)-2,4-pyrimidinediamine; 5-Fluoro-N4-(4-fluoro-3-methoxyphenyl)-N2-(indazolin-6-yl)-2,4-pyrimidinediamine; 5-Fluoro-N4-(4-fluoro-3-methoxyphenyl)-N2-(indazolin-5-yl)-2,4-pyrimidinediamine; N2-(3-Chloro-4-methoxy-5-methylphenyl)-5-fluoro-N4-{4H-imidazo[2,1-c]-benz[1,4]oxazin-8-yl}-2,4-pyrimidinediamine; N2-(3-Chloro-4-methoxy-5-methylphenyl)-5-fluoro-N4-(1-methylindazolin-6-yl)-2,4-pyrimidinediamine; N2-(3,5-Dimethoxyphenyl)-5-fluoro-N4-(1-methylindazolin-6-yl)-2,4-pyrimidinediamine; N2-(4-Chloro-2,5-dimethoxyphenyl)-5-fluoro-N4-(1-methylindazolin-6-yl)-2,4-pyrimidinediamine; N2-(3,5-Dimethoxyphenyl)-5-fluoro-N4-(indazolin-6-yl)-2,4-pyrimidinediamine; N2-(2,2-Dimethyl-3-oxo-4H-benz[1,4]oxazine-6-yl)-5-fluoro-N4-(1-methylindazolin-6-yl)-2,4-pyrimidinediamine; N4-(3-Chloro-4-fluorophenyl)-5-fluoro-N2-(1-methylindazolin-6-yl)-2,4-pyrimidinediamine; N4-(3-Chloro-4-fluorophenyl)-5-fluoro-N2-(1-methylindazolin-5-yl)-2,4-pyrimidinediamine; N4-(3-Chloro-4-fluorophenyl)-5-fluoro-N2-(indazolin-6-yl)-2,4-pyrimidinediamine; N4-(3-Chloro-4-fluorophenyl)-5-fluoro-N2-(indazolin-5-yl)-2,4-pyrimidinediamine; N4-(2,2-Dimethyl-3-oxo-4H-benz[1,4]oxazin-6-yl)-5-fluoro-N4-(indazolin-6-yl)-2,4-pyrimidinediamine Benzenesulfonic Acid Salt; N4-(2,2-Dimethyl-3-oxo-4H-benz[1,4]oxazin-6-yl)-5-fluoro-N4-(indazolin-6-yl)-2,4-pyrimidinediamine p-Toluenesulfonic Acid Salt; N4-(2,2-Dimethyl-3-oxo-4H-benz[1,4]oxazin-6-yl)-N2-[1-(2-ethoxycarbonylethyl)indazolin-5-yl]-5-fluoro-2,4-pyrimidinediamine; N4-(2,2-Dimethyl-3-oxo-4H-benz[1,4]oxazin-6-yl)-5-fluoro-N241-(3-hydroxypropyl)indazolin-5-yl]-2,4-pyrimidinediamine; N4-(2,2-Dimethyl-3-oxo-4H-benz[1,4]oxazin-6-yl)-5-fluoro-N2-{1-[2(N-methylaminocarbonyl)ethyl]indazolin-5-yl}-2,4-pyrimidinediamine; N4-(2,2-Dimethyl-3-oxo-4H-benz[1,4]oxazin-6-yl)-N2-[1-(2-ethoxycarbonylethyl)indazolin-6-yl]-5-fluoro-2,4-pyrimidinediamine; N4-(2,2-Dimethyl-3-oxo-4H-benz[1,4]oxazin-6-yl)-5-fluoro-N2-{1-[2-(N-methylaminocarbonyl)ethyl]indazolin-6-yl}-2,4-pyrimidinediamine; N4-(2,2-Dimethyl-3-oxo-4H-benz[1,4]oxazin-6-yl)-N241-(methoxycarbonyl)methyl-indazolin-5-yl]-5-fluoro-2,4-pyrimidinediamine; N4-(2,2-Dimethyl-3-oxo-4H-benz[1,4]oxazin-6-yl)-5-fluoro-N2-[1-(N-methylaminocarbonyl)methylindazolin-5-yl]-2,4-pyrimidinediamine; N2-(2,2-Dimethyl-3-oxo-4H-benz[1,4]oxazin-6-yl)-5-fluoro-N4-(indazolin-5-yl)-2,4-pyrimidinediamine; N2-(2,2-Dimethyl-3-oxo-4H-benz[1,4]oxazin-6-yl)-5-fluoro-N4-(1-methylindazolin-5-yl)-2,4-pyrimidinediamine; N2-(2,2-Dimethyl-3-oxo-4H-benz[1,4]oxazin-6-yl)-5-fluoro-N4-(indazolin-6-yl)-2,4-pyrimidinediamine; N4-(3,4-Dimethoxyphenyl)-5-fluoro-N2-(indazolin-5-yl)-2,4-pyrimidinediamine; N4-(2,2-Dimethyl-3-oxo-4H-benz[1,4]oxazin-6-yl)-5-fluoro-N2-[1-(2-methoxy-4-methoxycarbonylbenzyl)indazolin-6-yl)-2,4-pyrimidinediamine; N4-(3,4-Dichlorophenyl)-5-fluoro-N2-(1-methylindazolin-6-yl)-2,4-pyrimidinediamine p-Toluenesulfonic Acid Salt; N4-(3,4-Dichlorophenyl)-5-fluoro-N2-(2-methylindazolin-6-yl)-2,4-pyrimidinediamine; N4-(2,2-Dimethyl-3-oxo-4H-benz[1,4]oxazin-6-yl)-5-fluoro-N2-(2-methylindazolin-6-yl)-2,4-pyrimidinediamine; N4-(3-Chloro-4-methoxyphenyl)-5-fluoro-N2-(2-methylindazolin-6-yl)-2,4-pyrimidinediamine; N4-(3,4-Dichlorophenyl)-N242-(2-ethoxycarbonylethyl)indazolin-5-yl]-5-fluoro-2,4-pyrimidinediamine; N4-(2,2-Dimethyl-3-oxo-4H-benz[1,4]oxazin-6-yl)-N2-[2-(2-ethoxycarbonylethyl)indazolin-5-yl]-5-fluoro-2,4-pyrimidinediamine; 5-Fluoro-N4-(4-fluoro-3-methoxyphenyl)-N2-(1-methylindazolin-6-yl)-2,4-pyrimidinediamine; 5-Fluoro-N4-(4-fluoro-3-methoxyphenyl)-N2-(2-methylindazolin-6-yl)-2,4-pyrimidinediamine; N4-(3,4-Dichlorophenyl)-5-fluoro-N2-[1-(3-hydroxypropyl)indazolin-5-yl]-2,4-pyrimidinediamine Bis(p-Toluenesulfonic Acid Salt; N4-(3,4-Dichlorophenyl)-5-fluoro-N2-{2-[2-(N-methylaminocarbonyl)ethyl]indazolin-5-yl}-2,4-pyrimidinediamine; N4-(2,2-Dimethyl-3-oxo-4H-benz[1,4]oxazin-6-yl)-5-fluoro-N2-{2-[2(N-methylaminocarbonyl)ethyl]indazolin-5-yl}-2,4-pyrimidinediamine; N4-(3-Chloro-4-methoxyphenyl)-5-fluoro-N2-(1-methylindazolin-5-yl)-2,4-pyrimidinediamine; N4-(3-Chloro-4-methoxyphenyl)-5-fluoro-N2-(indazolin-5-yl)-2,4-pyrimidinediamine; N4-(3-Chloro-4-methoxyphenyl)-N241-(2-ethoxycarbonylethyl)indazolin-5-yl]-5-fluoro-2,4-pyrimidinediamine; N4-(3-Chloro-4-methoxyphenyl)-5-fluoro-N2-[1-(3-hydroxypropyl)indazolin-5-yl]-2,4-pyrimidinediamine; N4-(3-Chloro-4-methoxyphenyl)-5-fluoro-N2-{1-[2-(N-methylaminocarbonyl)ethyl]indazolin-5-yl}-2,4-pyrimidinediamine; and a salt, hydrate, solvate, or an N-oxide thereof.
 56. The method of claim 55, wherein the one or more additional compounds comprise methotrexate, an anti-TNF drug, or a combination thereof.
 57. The method of claim 56, comprising administering the compound of formula (I) and methotrexate.
 58. The method of claim 56, comprising administering the compound of formula (I) and one or more anti-TNF drugs.
 59. The method of claim 35, wherein the one or more additional compounds comprise methotrexate, an anti-TNF drug, or both.
 60. The method of claim 59, comprising administering the compound of formula (I) and methotrexate.
 61. The method of claim 59, comprising administering the compound of formula (I) and one or more anti-TNF drugs.
 62. The method of claim 41, wherein the one or more additional compounds comprise methotrexate, an anti-TNF drug, or both.
 63. The method of claim 62, comprising administering the compound of formula (I) and methotrexate.
 64. The method of claim 62, comprising administering the compound of formula (I) and one or more anti-TNF drugs.
 65. The method of claim 47, wherein the one or more additional compounds are selected from the group consisting methotrexate and anti-TNF drugs.
 66. The method of claim 65, comprising administering the compound of formula (I) and methotrexate.
 67. The method of claim 65, comprising administering the compound of formula (I) and one or more anti-TNF drugs.
 68. The method of claim 50, wherein the one or more additional compounds are selected from the group consisting methotrexate and anti-TNF drugs.
 69. The method of claim 68, comprising administering the compound of formula (I) and methotrexate.
 70. The method of claim 68, comprising administering the compound of formula (I) and one or more anti-TNF drugs. 